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Keywords:

  • afferent;
  • hypersensitivity;
  • inflammation;
  • jejunum;
  • visceral

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

Abstract  Background:  The mechanisms underlying endotoxin-induced hyperalgesia remain unknown. We aimed to study the mechanisms underlying the sensitizing action of lipopolysaccharide (LPS) on intestinal afferent responses to mechanical and chemical stimuli.

Methods:  Extracellular recordings of jejunal afferent nerve discharge were obtained from pentobarbitone-anaesthetized rats.

Results:  Lipopolysaccharide (6 mg kg−1, i.v.) stimulated a short-term, transient (<30 min) increase in chemosensitivity to systemic 5-HT (6 μg kg−1) and responses to mechanical distension and a delayed but maintained (>30 min) increase in spontaneous afferent discharge. Naproxen (10 mg kg−1) and the prostaglandin receptor antagonist AH6809 (1 mg kg−1) significantly attenuated both the short-term sensitization to mechanical distension and 5-HT and the long-term increase in baseline afferent firing following LPS. In contrast, the iNOS inhibitor aminoguanidine (15 mg kg−1) and the L-type calcium channel antagonist nifedipine (1 mg kg−1) both prolonged the period of afferent sensitization to distension and 5-HT without influencing the augmented baseline-firing rate. ω-Conotoxin GVIA attenuated the increase in afferent discharge to LPS, without any change in mechano- and chemosensitivity.

Conclusions:  The long-term (>30 min) increase in afferent firing following systemic LPS involves neurogenic release of prostanoids. The short-term (<30 min) sensitization also appears to depend on prostanoid release, while nitric oxide production may serve to down-regulate LPS-induced afferent hypersensitivity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

The gastrointestinal tract (GI) must balance two ostensibly conflicting tasks. On the one hand it must absorb nutrients, water and electrolytes essential for continued metabolism and on the other must protect against potentially harmful bacteria pathogens, toxins and antigens. The adaptations that favour the former are a defence liability for the latter. Therefore an elaborate gastrointestinal immune system has evolved to protect the organisms from potential harmful agents that cross the gastrointestinal mucosa. Part of this protective mechanism is an innate sensitivity to harmful bacteria.1 Recognition of the cellular components of harmful bacteria generates a macrophage-driven cytokine cascade that is referred to as an acute phase response and drives a local inflammatory reaction also generating behavioural responses known as sickness behaviour that include fever, anorexia and hyperalgesia.2, 3 Lipopolysaccharide (LPS) from gram negative bacteria is used experimentally to trigger an acute phase response including hyperalgesia.4–6 High doses of LPS are lethal and protection from lethality has been used as an experimental model to investigate modulation of immune responses.7 Clinically this is equivalent to the events that follow breakdown in the gastrointestinal protective mechanisms when large numbers of bacteria translocate the intestinal mucosa. One dramatic example of when this occurs is during multi-organ failure in critical care patients. Major trauma with hypoperfusion of the GI tract leads to a breakdown in the protective mechanisms and bacteria cross the mucosa epithelium triggering a major systemic inflammatory response.8

The macrophage-driven cytokine cascade following LPS administration gives rise to an increase in circulation IL-1β and TNF-α. These cytokines orchestrate both the local inflammatory response and the CNS consequences that are manifested as illness behaviour.3 These CNS consequences appear to involve both direct effects of circulating cytokine and activation of afferent inputs to the central circuits that regulate temperature, feeding behaviour and pain modulation.9 Thus brain fos expression following LPS or IL-1β have been shown to be attenuated following procedures that interrupt the afferent traffic to the brainstem and spinal cord in the vagus and spinal nerves, respectively,10, 11 although this finding remains controversial.12 Such observation suggests that afferent neuron excitability is augmented following treatment with LPS and a number of studies have investigated modulation of afferent sensitivity in response to cytokines such as IL-1β.13, 14 However, the mechanism underlying any change in afferent sensitivity have not been systematically investigated. The aim of the present study, therefore, was to investigate the effects of LPS on afferent nerve discharge emanating from the GI tract and to probe the mechanisms that underlie changes in afferent sensitivity to mechanical and chemical stimuli.

Animal preparation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

Experiments were conducted on male Wister rats (300–400 g). The animals were fasted for 12 h before the experiments but were allowed free access to water. The institutional guidelines for the use and care of laboratory animals were followed throughout the study. General anaesthesia was produced by an intraperitoneal injection of pentobarbitone sodium (60 mg kg−1) and was sustained by intravenous infusion (0.5–1 mg kg−1 min−1) of the anaesthetic. The trachea was intubated with a short length of tubing to facilitate spontaneous respiration. The right external jugular vein was cannulated with two saline-filled cannula to facilitate maintenance anaesthesia and systemic administration of drugs. The left common carotid artery was cannulated to record arterial blood pressure (Transducer: DT-XX, Ohmeda Pte Ltd., Singapore; Amplifier: Neurolog Pressure Amplifier NL108, Digitimer Ltd, Welwyn Gerden City, UK). Body temperature was monitored with a rectal thermometer and maintained at around 37 °C by means of a heated table. A midline laparotomy was performed and the cecum was excised. A 10-cm loop of proximal jejunum was isolated and cannulated at the oral and anal ends. The oral cannula served as a drain and remained open in the time-course studies described subsequently, in order to minimise any changes in intraluminal pressure arising as a consequence of motor activity or secretion into the loop. In other experiments, this oral cannula was connected to a pump-driven syringe to deliver saline into the loop of jejunum at a constant rate of 1 mL min−1 up to a distending pressure of 60 cm H2O. The latter was monitored by connecting the arboral cannula to a pressure transducer (Neurolog Pressure Amplifier NL 108, Digitimer Ltd). The abdominal incision was sutured to a 5-cm diameter steel ring to form a well that was subsequently filled with prewarmed (37 °C) light liquid paraffin.

Nerve preparation and afferent recording

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

A single mesenteric arcade 5–7 cm distal to the ligament of Treitz was placed on a black perspex platform to enable one of the paravascular nerve bundles to be dissected from the surrounding tissue. The nerve bundle was severed distal from the wall of the jejunum (approximately 1–1.5 cm) to eliminate efferent nerve activity. It was then attached to one of a pair of platinum electrodes with a strand of connected tissue wrapped around the other to act as a differential. The electrodes were connected to a Neurolog Headstage (Neurolog NL 100) and the signal from them was amplified (Neurolog NL 104) and filtered (Neurolog NL 125). The neurogram was displayed on a storage oscilloscope (TDS 310; Tektronix, Cologne, Germany) and relayed together with the signals from the arterial and intra-jejunal pressure transducers into a 1401 plus interface [Cambridge Electronic Design (CED), Cambridge, UK]. A PC running spike 2 software sampled these signals online.

Protocol during mesenteric afferent recording

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

After preparation of a mesenteric afferent nerve bundle for electrophysiological recording, a 30-min period of baseline activity was recorded prior to any experimental protocols. Two different types of experimental protocol were then followed.

Time-course studies  Lipopolysaccharide from Escherichia coli (026 : B6) was infused intravenously (6 mg kg−1) over a period of approximately 3 min. Control animals received the saline vehicle. Cardiovascular parameters and the mesenteric afferent discharge were monitored for 120 min after injection of LPS. Subsequent experiments were designed to determine the mechanism underlying these changes following LPS and in these experiments pharmacological agents or their appropriate vehicle were administered intravenously 10 min prior to the injection of LPS.

Effect of LPS on mechanosensitivity and responses to 5-HT  These experiments followed a similar time course to those described above except that distension up to an intraluminal pressure of 60 cm H2O and 5-HT (6 μg, i.v.) were administered 10 min before and 5 min after the administration of pharmacological agents. LPS was given 10 min after drugs were administered. Then, intraluminal distension and 5-HT were repeated at 15-min intervals. A schematic view of the protocol is given in Figure 1. The afferent nerve responses before administration of drugs are referred to as ‘baseline responses’.

image

Figure 1. Schematic view of the experimental protocol for the investigation of antagonists.

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Drug treatments  Animals were divided randomly into five drug treatment groups (n ≥ 4) receiving either naproxen (10 mg kg−1), AH6809 (1 mg kg−1), aminoguanidine (15 mg kg1), nifedipine (1 mg kg−1), ω-conotoxin (30 μg kg−1) or a saline and DMSO vehicle-treated groups. All drugs were given in a single commonly administered dose as the complexity of the experimental approach deemed not to be feasible for dose–response studies. The doses were chosen according to previous investigations from our own group and others: naproxen,15 AH 6809,16 aminoguanidine,17 nifedipine,16, 18 and ω-conotoxin.18 The half-lives of the following drugs administered were assumed to exceed the observation period based on a review of the literature: naproxen,19 aminoguanidine,20 and nifedipine.21 During our own previous experimental work,16, 18 we have still observed effects of AH 6809 and conotoxin more than 90 min after administration of these drugs (unpubl. obs.). These drugs altered neither the baseline afferent nerve response nor the afferent nerve response to ramp distension or 5-HT.

Analysis of data

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

Afferent neurograms were analysed using spike 2 software (CED) in order to count the total number of action potentials crossing a preset threshold in sequential time bins. The mean baseline values for each of the measured variables of afferent activity (in spikes s−1) intrajejunal pressure (in cm H2O) and blood pressure (in mmHg) were determined over a 30-s period prior to any treatment. For the time-course studies these parameters were assessed at various time points from 30 s to 120 min after administration of LPS. At each time point the mean discharge rate and mean arterial blood pressure were determined over a 10-s period. Responses to distension were determined by quantifying the firing frequency over a 3-s period at 10 cm H2O increments in intrajejunal pressure. The baseline firing prior to distension was subtracted in order to provide values for the increase in discharge in response to distension. The afferent response to 5-HT was similarly obtained from the peak-firing rate (over a 3-s period) minus baseline discharge. Data are presented as the arithmetic mean ± SEM from four or more animals per vehicle or antagonist-treated or untreated group. Where n values are given they refer to the number of animals. Significant differences between group means were determined using one-way anova followed by Dunnett's test. A probability of P < 0.05 was considered to be indicative of statistically significant difference. In some experiments single unit analysis of the whole mesenteric nerve discharge was performed using the template matching algorithm in spike 2 (CED) as described previously.22

Drugs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

Lipopolysaccharide (E. coli 026 : B6), aminoguanidine hemisulphate, nifedipine, naproxen and AH6809 were purchased from Sigma (Munich, Germany). ω-Conotoxin GVIA was obtained from Alomone Laboratories, Jerusalem, Israel. Apart from nifedipine and AH6809 all of the compounds were each dissolved in 0.09% w/v sodium chloride solution (saline) frozen in aliquots that were thawed when required. Nifedipine and AH6809 were dissolved in 25% w/v diamethyl sulfoxide in saline on the day of the experiment and thereafter protected from light.

Time course of the cardiovascular and mesenteric afferent response to LPS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

Lipopolysaccharide caused a marked biphasic fall in arterial blood pressure and a slowly developing, maintained increase in whole nerve mesenteric afferent discharge (Fig. 2). The first rapid phase of the cardiovascular response occurred approximately 2.5 min following LPS administration and reached a nadir after 3.5–5 min (93 ± 8.5 mmHg vs 124 ± 2.9 mmHg at baseline, P < 0.05, n = 10, Fig. 3A). This depressor effect rapidly recovered so that blood pressure was not different from baseline between 25 and 40 min after LPS. Thereafter, a second prolonged decrease in blood pressure reached a nadir of 98.2 ± 5.5 mmHg 70 min after LPS (P < 0.05; Fig. 3A). Blood pressure was constant throughout the 2-h recording period in animals treated with vehicle.

image

Figure 2. Representative example of the cardiovascular and mesenteric afferent response to systemic lipopolysaccharide (LPS) (6 mg kg−1, i.v.). Upper trace is the arterial blood pressure showing a marked biphasic fall in blood pressure. Below is a sequential rate histogram of whole nerve mesenteric afferent discharge showing a sustained increase in discharge frequency following LPS. Snapshots taken from the raw neurogram at the times indicated illustrate the increased afferent firing of action potentials after LPS together with recruitment of larger amplitude spikes.

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image

Figure 3. Time course of the blood pressure response to lipopolysaccharide (LPS) (6 mg kg−1, i.v.). Panel (A) shows the mean data for mean arterial blood pressure for the 2-h period following administration of LPS or vehicle. The biphasic nature of the hypotension is clearly illustrated with # (P < 0.05) and ## (P < 0.01) indicating the time points at which the blood pressure was significantly different between the two groups. In order to quantify the various components of this response, mean blood pressure at 5 min were taken to represent the initial transient component of the response, 30 min represents the period when blood pressure had returned to baseline values and 60 min represents the sustained second component. This data is shown in the histograms in panel (B) expressed as a % of the baseline blood pressure prior to administration of LPS. The first column in each histogram is the data from panel (A) expressed as a %. Subsequent columns represent the data taken from animals after the various pharmacological treatments shown in the legend. Note that aminoguanidine (15 mg kg−1) significantly attenuated the first component of the blood pressure fall in response to LPS while ω-conotoxin GVIA (30 μg kg−1) abolished the sustained second phase of the blood pressure response. All other treatments were without effect on the blood pressure response despite marked influences on the mesenteric afferent response to LPS (see later) with the exception of naproxen, which enhanced the magnitude of the first component of the blood pressure response.

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There was a small transient increase in afferent firing during the initial transient fall in arterial blood pressure, increasing from a baseline value of 19.1 ± 2.2 to 33.2 ± 7.4 impulses s−1 (imp s−1) 5 min following LPS (n = 5, Fig. 4A). However, afferent discharge started to increase most dramatically during the period when arterial blood pressure had recovered to baseline and continued to increase to reach a maximum firing rate of 76.9 ± 12.4 imp s−1, 100 min after treatment with LPS. From the raw nerve trace it was observed that this increase in afferent firing was because of both an increased discharge frequency of spontaneously active units and the recruitment of new large amplitude spikes. In vehicle-treated animals, afferent nerve discharge remained stable at baseline levels throughout the 120-min recording period.

image

Figure 4. Time course of the whole nerve mesenteric afferent response to lipopolysaccharide (LPS) (6 mg kg−1, i.v.). Panel (A) shows the mean data for afferent firing for the 2-h period following administration of LPS or vehicle. The sustained increase in afferent firing is clearly illustrated with # (P < 0.05) and ## (P < 0.01) illustrating the time point at which afferent discharge was significantly increased in comparison to vehicle. Data at 5, 30 and 60 min after LPS were quantified as % increase above baseline and are shown in the histograms in panel (B). The first column in each histogram is the data taken from panel (A). Subsequent columns represent the data taken from animals after the various pharmacological treatments shown in the legend. Note that naproxen (1 mg kg−1), AH-6809 (1 mg kg−1) and ω-conotoxin GVIA (30 μg kg−1) each significantly attenuated the augmented afferent firing in response to LPS.

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Pharmacology of the cardiovascular response  As the nature of the cardiovascular response was biphasic, time points were defined at 5, 30 and 60 min which corresponded to the first phase, recovery and second phase of the cardiovascular response profile (Fig. 3A,B). Naproxen (10 mg kg−1, i.v.) had no effect on baseline blood pressure (122.1 ± 4.7 mmHg, vs 121.5 ± 3.9 mmHg, P > 0.05, n = 10) and the cardiovascular response to LPS was similar to that in the vehicle-treated control group except that the initial fall in blood pressure at 5 min was somewhat exaggerated after LPS.

The cardiovascular response to LPS was not different following pretreatment with AH-6809 compared to controls. The systemic arterial blood pressure was 148.5 ± 2.6 mmHg following pretreatment with the prostaglandin receptor antagonist AH-6809 (1 mg kg−1, i.v.) and 142.2 ± 2.9 mmHg following its vehicle (25% DMSO, 1 mL kg−1, i.v.).

Aminoguanidine (15 mg kg−1) increased arterial blood pressure from 126 ± 2.4 to 138.3 ± 3 mmHg (P < 0.05, n = 5). More strikingly, the initial depressor effect following LPS was attenuated (P < 0.05), while the late depressor response was unchanged by aminoguindine.

Nifedipine (1 mg kg−1, i.v.) reduced arterial blood pressure from 125.9 ± 4.3 to 95.5 ± 4.45 mmHg (P < 0.05), while the cardiovascular response to LPS was not altered by nifedipine.

ω-Conotoxin (30 μg kg−1, i.v.) reduced arterial blood pressure from 118.1 ± 6.4 to 84 ± 5.8 mmHg (P < 0.05, n = 5). The early depressor effect of LPS was unchanged by ω-conotoxin but the second, maintained fall in blood pressure was significantly attenuated and indeed became significantly elevated above baseline parameters (94.9 ± 3.5 mmHg, at 40 min, P < 0.05).

Pharmacology of the afferent nerve response  Data on baseline afferent firing rates before and after naproxen and the other antagonists are given in Table 1 (Fig. 4B). Naproxen (10 mg kg−1, i.v.) had no effect on the baseline afferent firing rate. However, the increase in afferent nerve discharge following LPS was attenuated following naproxen treatment during the late phase of the response (P < 0.05 at 60 min).

Table 1.  Afferent discharge at baseline and following normal saline (vehicle) or different antagonists. Note that the impulse frequencies did not differ in the various subgroups before and after the different pretreatments
AntagonistBefore pretreatment (mean ± SEM)After pretreatment (mean ± SEM) P-value
Normal saline (vehicle)20.2 ± 2.519.1 ± 2.20.795
Naproxen19.7 ± 3.318.1 ± 3.30.747
AH-680920.9 ± 1.422.8 ± 1.30.356
Aminoguanidine22.3 ± 1.320.8 ± 0.80.328
Nifedipine23.5 ± 3.323.9 ± 2.10.924
Conotoxin23.0 ± 2.924.7 ± 1.60.630

As observed for naproxen, AH-6809 (1 mg kg−1, i.v.) did not change afferent discharge at baseline but the afferent nerve response to LPS was attenuated at 60 min (P < 0.05).

In contrast to the modulation of the cardiovascular response, the increase in afferent nerve discharge to LPS following pretreatment with Aminoguanidine (15 mg kg−1) was comparable to control.

While the afferent nerve response to LPS was unchanged in animals receiving the L-type calcium channel antagonist Nifedipine (1 mg kg−1, i.v.), afferent discharge was attenuated following pretreatment with the N-type calcium channel antagonist ω-conotoxin, especially during the later time points (P < 0.05).

Response to 5-HT following systemic LPS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

5-HT (6 μg, i.v.) evoked a marked but transient increase in mesenteric afferent firing (Fig. 5B). In vehicle-treated animals this response to 5-HT was well maintained over the 2-h time course of these experiments. After LPS however, the magnitude of the response to 5-HT was augmented 15 and 30 min after treatment (Fig. 5B).

image

Figure 5. Sensitivity of mesenteric afferent bundles to 5-HT. Panel (A) shows an example of a mesenteric afferent bundle responding to systemic administration of 5-HT. Afferent firing increased dramatically peaking within seconds of administration. The response to 5-HT over time is illustrated in panel (B) which shows the absolute peak increase in afferent discharge over baseline before and after lipopolysaccharide (LPS) or vehicle. Note that the response to 5-HT is well maintained over time after vehicle treatment but is significantly increased 15 and 30 min after LPS (P < 0.05). The influence of pharmacological treatments on the time course of hypersensitivity to 5-HT is shown in panel (C). The first column is the data from panel (B). Subsequent columns represent the data taken from animals after the various pharmacological treatments shown in the legend. Note that naproxen and AH-6809 attenuate the response to 5-HT at 15 and 30 min. In contrast aminoguanidine and nifedipine increase the magnitude of, and extend the duration of, the LPS-mediated sensitization of the response to 5-HT.

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Both naproxen (10 mg kg−1, i.v.) and AH6809 (1 mg kg−1, i.v.) prevented the sensitization of the 5-HT response following LPS treatment. In contrast the response to 5-HT was increased following aminoguanidine at 15, 30 and 90 min and for nifedipine only at 90 min after LPS administration. ω-Conotoxin had no effect on the afferent response to 5-HT following LPS (Fig. 5C).

Mechanosensitivity following systemic LPS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

During ramp saline distension there was a pressure dependent increase in whole nerve intestinal afferent discharge (Fig. 6A). In vehicle-treated animals the afferent response to distension were consistent over time. However, in animals treated with LPS there was a significant increase in mechanosensitivity at distending pressures exceeding 20 cm H2O and was greatest at 60 cm H2O (Fig. 6B). However, this increased mechanosensitivity was short lived, being significantly elevated 15 min following LPS but comparable to control when the distension protocol was repeated 15 min later (Fig. 6C).

image

Figure 6. The effect of lipopolysaccharide (LPS) on mechanosensitivity of mesenteric afferents. Panel (A) shows a representative example of the intrajejunal pressure (top trace) and afferent discharge (below) during ramp distension of the jejunal segment with isotonic saline (1 mL min−1 to 60 cm H2O) 15 min before and 15 and 30 min after treatment with LPS. Note that afferent discharge increased dramatically during distension and that this increase was augmented 15 min after LPS but not after 30 min. Panel (B) shows the stimulus–response function of mesenteric afferent responses to distension by plotting the increase in whole nerve firing above baseline against intraluminal pressure at each of the three time points illustrated in (A). Note that at pressure >20 cm H20 the response to distension is significantly elevated 15 min after LPS but has returned to control levels at 30 min. The time course of mechanical hypersensitivity is shown in panel (C) where the increase in afferent discharge at 60 cm H2O expressed as a % of that prior to treatment is plotted for the 2-h period following LPS or vehicle. Note that after vehicle the response to distension is unchanged over time. However, LPS significantly increases the response after 15 min but this has recovered at 30 min and remains unchanged for the remainder of the experimental period.

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Thirty-nine distension-sensitive single afferent units were discriminated from these control experiments with LPS (Fig. 7). Among them 11 units (28.2%) were defined as low threshold afferents as they reached maximal discharge rates at intraluminal pressures below 20 cm H2O. Sixteen single units (41%) were discriminated with thresholds for activation above 20 cm H2O and these are referred to as high threshold afferents. The remaining 12 single units (30.8%) had thresholds <20 mmHg but increased their discharge rate over the entire range of intraluminal distension pressures up to 60 cm H2O, these are considered as wide dynamic range afferents. The sensitivity of these afferent populations were differentially influenced 15 min after treatment with LPS. Both the high threshold and wide dynamic range sensitive populations had increased mechanosensitivity. In contrast the low threshold mechanosensitive populations were not sensitized by LPS. Indeed the discharge rate of the low threshold population at 10 cm H2O pressure was significantly attenuated 15 min after LPS administration compared to the control response before LPS was administered.

image

Figure 7. Stimulus–response function of mesenteric mechanosensitive afferents. Single unit analysis was used to discriminate afferents with low and high thresholds for activation and afferents that responded over a wide-dynamic range (see text for definitions). A representative analysis with discriminated single units and the corresponding rate histograms is shown in the upper left panel. The upper right panel shows the mean response profile of 11 low threshold afferents whose firing increased dramatically upon distension and plateaued at pressures <20 cm H2O. Following lipopolysaccharide (LPS) the response of these afferents to distension was attenuated and this was significant at 10 cm H20. The left lower panel shows that firing of 16 afferents with high thresholds for activation was markedly augmented after LPS. The sensitivity of wide-dynamic range afferents (right lower panel) was also increased following LPS.

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The stimulus–response profile to ramp distension was attenuated in animals treated with naproxen. The increase in whole nerve firing rate at 60 cm H2O intraluminal pressure observed after LPS was significantly attenuated over the entire 2-h time course of these experiments. At the 15 min time point, the transient sensitization was eliminated and an afferent nerve response to distension was observed in naproxen pretreated animals that was below the level of the response in control animals before LPS administration.

The prostaglandin receptor antagonist AH-6809 abolished the LPS induced transient sensitization to distension at high pressure normally observed at 15 min post-LPS but had no effect at subsequent time points. Aminoguanidine prolonged the time course of the augmented mechanosensitivity in response to LPS. The sensitization of the distension response at 60 cm H2O that occurred only at 15 min in control animals persisted at 30 min after LPS in animals treated with aminoguanidine.

Nifedipine extended the duration of sensitization beyond the 15 min seen in control animals to 90 min after treatment with LPS. ω-Conotoxin had no effect on the afferent response to distension over the time course of these experiments (Fig. 8).

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Figure 8. Pharmacological manipulation of afferent sensitivity to distension. This histogram illustrates the sensitivity of mesenteric afferents to distension over time following lipopolysaccharide (LPS). The first column is the data from Fig. 6C showing the afferent firing after LPS as a % of the control response before treatment. Subsequent columns represent the data taken from animals after the various pharmacological treatments shown in the legend. Note that naproxen attenuates the response to distension throughout to a level below the control response. In contrast AH-6809 prevents the sensitized response to distension at 15 min, while responses at 30 and 90 min are not different from those control animals. Following treatment with aminoguanidine and nifedipine the duration of sensitization is extended beyond 15–30 min in the case of aminoguanidine and 90 min after nifedipine.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References

Systemic LPS triggered both a decrease in arterial blood pressure and a potent intestinal afferent nerve response. The blood pressure decrease was bi-phasic with an initial transient rapid component followed by a long-term hypotension, which began about 40 min after LPS and was maintained for the remainder of the study. Intestinal afferent nerve discharge started to increase 5 min following LPS administration and reached a plateau after about 60 min with an approximately threefold increase in afferent discharge rate above baseline. Mechanosensitivity to distension and the chemosensitive afferent response to 5-HT were also augmented by LPS but the time course of these changes were different from that of the baseline-firing rate. Response to distension at 5-HT were augmented 15 min after LPS but had returned to control levels at the time the baseline firing rate was beginning its dramatic increase. Both naproxen (10 mg kg−1, i.v.) and AH6809 (1 mg kg−1, i.v.) attenuated the sensitization of the response to 5-HT and mechanical distension following LPS treatment, while the magnitude and the duration of sensitization to both stimuli were increased by treatment with aminoguanidine and nifedipine.

The cardiovascular response to LPS has been extensively investigated. A major determinant of the hypotensive response appears to depend upon the increased expression of the inducible isoform of nitric oxide synthase and subsequent release of this highly potent vasodilator agent.23 However, what is surprising about the current study is that the effect of aminoguanidine, which at the dose used is considered selective for iNOS in the rat,24 was restricted to just the first phase of the fall in arterial blood pressure. This isoform of NOS is not constitutively expressed yet the aminoguanidine-sensitive component of the cardiovascular response peaked after only 3.5 min. It seems extremely unlikely that iNOS was expressed within this short time interval. One potential explanation is that aminoguanidine may also have influenced the constitutive forms of NOS. Alternatively, iNOS may already have been upregulated secondary to bowel manipulation during the 1-h preparation prior to recordings.25 Aminoguanidine would then inhibit this expressed iNOS which, in controls, may have triggered nitric-oxide (NO) release with the mentioned effect on the early phase of the blood pressure response to LPS. Contrary to the early phase, the later hypotension appears to be independent of treatment with aminoguanidine indicating that NO does not appear to contribute to the late phase hypotensive response, an observation that has been described previously.26 The late phase hypotension, however, was attenuated by treatment with the N-type calcium channel blocker ω-conotoxin. Indeed after treatment with this agent there was an increase in blood pressure during the later phase of the response to LPS. This implies that the arterial blood pressure reflects a balance of vasodilator and vasoconstricting factors with the former dominating in the absence of pharmacological treatment. However, after attenuating cardiovascular reflexes following treatment with ω-conotoxin the vasoconstrictor mechanisms become unmasked. This observation indicates that a neural reflex-driven vasodilation contributes to the later phase cardiovascular response to LPS. The trigger for this reflex vasodilation is not apparent but since it is only manifested in the later phase of the response to LPS this implies that the build up of inflammatory mediators as part of the cytokine cascade are the likely cause.

The increase in mesenteric afferent discharge followed a similar time course to the delayed hypotensive response and like the latter was attenuated by ω-conotoxin. A likely explanation for the correlation between blood pressure and mesenteric afferent firing is that ω-conotoxin may have attenuated the LPS-driven inflammatory response as have been previously described27 with a subsequent reduced release of inflammatory mediators that are responsible for the hypotensive and afferent nerve response. Alternatively, this correlation may imply that the two events are linked and that the fall in blood pressure is the trigger for the change in mesenteric afferent firing. Indeed these afferents are known to be sensitive to ischemia and thus inadequate mesenteric perfusion during major falls in blood pressure may indeed give rise to an increase in afferent firing.28 However, this is an unlikely explanation for a number of different reasons. Firstly, in experiments in which the blood pressure was deliberately manipulated by infusion of sodium nitroprusside, the blood pressure needed to fall to approximately 50 mmHg before any impact on afferent firing was observed.29 In the present study, the mean blood pressure during this delayed phase in hypotension was between 90 and 100 mmHg, considerably higher than this threshold. Secondly, the most dramatic fall in blood pressure occurred early following administration of LPS and this nadir in blood pressure was associated with a minimal increase in afferent firing compared to that observed later in the response. Thus when the cardiovascular response is taken as a whole the afferent firing did not mirror these changes in blood pressure.

The mechanosensitivity of afferent nerves within these mesenteric bundles raises the possibility that intestinal motor events might also contribute to the altered afferent firing. In other words, the afferent response may be secondary to changes in intestinal motility. This indeed appeared to be the case in preliminary experiments30 in which, with the intestinal loop closed, there was an increase in intraluminal pressure in response to LPS associated with both an increase in muscle tone and distension arising from the secretion of fluid into the loop. In this preliminary study it was also shown that treatment with nifedipine attenuated these motor and secretor events and also attenuated the increase in whole nerve afferent firing. However, in the present experiments the intestinal loop remained open so that any increases in intraluminal pressure arising from motility or secretion would be attenuated. Indeed the pressure within these intestinal loops with the drain open failed to register any changes in pressure. It appears unlikely, therefore, that the afferent response is secondary to changes in motility or secretion and consistent with this view is the observation that in these experiments nifedipine had no effect on the increase in afferent firing following LPS.

These observations lead us to conclude that the increase in afferent firing following LPS occurs following direct stimulation of the afferent nerve endings by mediators released during the microphage-driven cytokine cascade. Our group and others have shown that a variety of mediators contribute to sensitisation of the afferent nerve endings and these mediators include amines, purines, prostanoids, kinines, etc.29 However, in the sensitisation process prostanoids appear to play a pivotal role having a direct effect on afferent sensitivity and potentiating the effect of other mediators.16, 31, 32 In the present study we therefore focused on the role of prostanoids in the LPS mediated increase in afferent firing. Moreover it is well established that an isoform of cyclo-oxygenase (COX2) is elevated during gastrointestinal inflammation.33 and that changes in COX expression can occur in intestinal macrophages following treatment with LPS.34 In the present study, therefore, we utilised naproxen, a non-specific blocker of cyclo-oxygenases, in order to investigate the contribution of prostanoids to the increase in afferent firing. Treatment with naproxen had no effect on baseline afferent firing but markedly attenuated the increase in afferent discharge following LPS. Thus prostanoids would seem to be implicated in the increased sensitivity of afferent firing during LPS administration although which of the COX isoforms mediating this effect remain to be determined. Treatment with the prostaglandin receptor antagonist AH6809 also attenuated the response to LPS implying that generation of prostaglandins of the E series contribute to the increased afferent sensitivity. This observation is consistent with recent studies showing that sensitisation of esophageal afferents can be attenuated by blocking the EP-1 receptor.35 There are varying reports on the specificity of AH 6809 as regards the different prostaglandin EP receptor subtypes.36 As we did not perform dose–response experiments in the present study, it is virtually not possible to determine from our data which of the EP receptor subtypes was involved. However, from a previous study it appears that prostaglandin E2 acts on intestinal afferents in the rat mainly via the EP-1 receptor.16

The effect of LPS on mechanosensitivity was investigated by comparing the response to ramp distension up to 60 cm H2O before and after treatment with LPS. Indeed LPS was found to increase the discharge rate at distending pressures >20 cm H2O although this increase in sensitivity was short lived. Mechanical sensitisation was present 15 min after LPS but had returned to baseline 15 min later. This is intriguing for two reasons. Firstly, the sensitisation at 15 min occurs prior to any overall increase in whole nerve baseline afferent firing to LPS. Secondly, mechanosensitivity has returned to normal when the major change in baseline afferent firing following LPS occurs. This would suggest that different mechanisms are responsible for the mechanical hypersensitivity and the increase in baseline afferent discharge. Another intriguing aspect of this finding is that it is only mechanosensitivity to higher distending pressures that is augmented by LPS (Fig. 7). This was apparent following single unit analysis of the stimulus–response profiles of individual afferent fibres within the whole nerve mesenteric bundles, which confirmed that only afferents sensitive to higher distending pressures were influenced by treatment with LPS. Mechanosensitive afferents fall into three categories according to their stimulus–response profile. Low threshold afferents respond to distensions within the physiological range, i.e. they have low thresholds for activation and peak responses at levels of distension that are physiologically encountered within the gastrointestinal tract. In contrast high threshold mechanoreceptors respond only to levels of distension that exceed the physiological range and continue to encode pressures above this. The third category of afferents are wide dynamic range afferents that have a low threshold for activation but continue to encode levels of distension into, and above, the physiological range.37 In the present study, only wide dynamic range and high threshold mechanoreceptors were sensitised by treatment with LPS. Their relative proportion in the mesenteric bundle was obviously substantial as the sensitization of mechanical distension following LPS was still present in the whole nerve response, although the relative contribution of the different mechanosensitive subpopulations cannot be adequately studied with our preparation. Indeed the low threshold mechanoreceptors showed signs of an attenuated response profile after LPS. These different afferent stimulus–response functions correlate closely with the projection pattern of the afferent fibres present within the mesenteric nerve bundles. Low threshold mechanoreceptors appear to project predominantly via vagal pathways to the brainstem while high threshold and wide dynamic range fibres project mainly in splanchnic nerves to the spinal cord.38 The augmented afferent response in the high and wide dynamic range fibres would imply that it is spinal afferents that are sensitised by LPS and not vagal afferents.

The augmented sensitivity to mechanical stimulation 15 min after LPS appears to depend upon prostanoids since it is markedly attenuated by treatment with both naproxen, to block prostanoid production, and AH6809 to block prostaglandin receptors (Fig. 8). However, an interesting observation is that treatment with aminoguanidine and nifedipine extended the time course of sensitisation to distension. Thus after aminoguanidine, hypersensitivity to distension was present after 30 min and in the case of nifedipine up to 90 min following treatment with LPS. This observation implies that the recovery of mechanosensitivity observed in untreated animals arose from an active process which down-regulated mechanosensitivity. Thus both stimulation and inhibition of afferent sensitivity appear to be at play in determining the response to mechanical stimulation. These inhibitory influences may be at play during low levels of distension and might account for the attenuated response of low threshold mechanoreceptors to 10 cm H2O distension in control animal and the augmentation of the low threshold component to the whole nerve response following treatment with aminoguanidine and nifedipine. The effect of aminoguanidine would imply nitric oxide as one such inhibitory factor. Consistent with this view is the observation that mechanosensitivity of isolated DRG neurons in culture is increased after blocking NOS with NO acting to inhibit voltage-gated sodium and calcium channels.39 Alternatively, nitric oxide may influence the release of other mediators from other cellular sources. These cellular sources are likely to include macrophages, which are the primary target for LPS, and which release NO and TNFα via an L-type calcium channel dependent mechanisms.40, 41 Such effects may contribute to the observation that nifedipine augments and prolongs mechanosensitivity in the present study. What is perhaps surprising is that aminoguanidine and naproxen have no effect on the baseline increase in afferent firing. This might indicate that the population of afferents that contributing to baseline firing are different from those that are sensitised to mechanical stimulation and it might also imply that different mediators give rise to the two events. These inflammatory mediators may be generated within the gastrointestinal wall itself or generated elsewhere, e.g. liver and lungs and delivered to the gut via the systemic circulation. In either event clearly these mediators have a profound influence on the sensitivity of mesenteric afferent nerve fibres.

Another mechanosensitive afferent population present within these mesenteric nerve bundles are the intestinofugal fibres that project from the myenteric plexus to the prevertebral ganglia and which take part in local inhibitory reflexes.42 These intestinofugal fibres receive a pronounced synaptic input from sensory neurones within the enteric nervous system and as such the firing of these intestinofugal fibres is determined by synaptic input within the enteric network.42 The observation that ω-conotoxin, which would attenuate this synaptic actually has no effect on the mechanosensitivity of these afferent bundles or the changes in sensitivity following LPS treatment would imply that intestinofugal fibres do not contribute in a major way to the response profiles described in the present study.

The sensitivity of mesenteric afferent nerve bundles to 5-HT has been described in detail previously.18, 43 The afferent nerve response to 5-HT appeared to be influenced by mediators released following treatment with LPS in a manner similar to that described for changes in mechanosensitivity. In particular, the magnitude of the response was attenuated by interfering with prostanoid production or prostaglandin receptors. Also the duration of the sensitisation was extended by treatment with aminoguanidine and nifedipine. The major effect of 5-HT on mesenteric afferent firing in this experimental model is via 5-HT3 receptors present on the terminals of vago-mucosal afferents.22 While the contribution of 5-HT3 receptors to the present sensitisation was not investigated it appears likely that the augmented response to 5-HT arises from augmented firing of vagal chemosensitive afferents. The implication then is that mediators released in response to systemic LPS have profound influence on both mechano and chemosensitivity of afferent fibres originating from the bowel wall.

In conclusion, a differential modulation of afferent sensitivity and arterial blood pressure was observed following systemic LPS and this involves both prostanoids and NO-dependent mechanisms. Both vagal and spinal afferents appear to be modulated and a complex interplay between factors that increase and factors that decrease intestinal sensitivity are at play. This modulation of intestinal afferent sensitivity during inflammation may have implications for the hypersensitivity concept of irritable bowel syndrome.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Animal preparation
  6. Nerve preparation and afferent recording
  7. Protocol during mesenteric afferent recording
  8. Analysis of data
  9. Drugs
  10. Results
  11. Time course of the cardiovascular and mesenteric afferent response to LPS
  12. Response to 5-HT following systemic LPS
  13. Mechanosensitivity following systemic LPS
  14. Discussion
  15. Acknowledgment
  16. References
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