Neuro-Gastroenterology and Nutrition Unit, Institut National de la Recherche Agronomique, 31931 Toulouse, France
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ABSTRACT |
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Intraperitoneal
lipopolysaccharide (LPS) produces somatic hyperalgesia, releases
interleukin (IL)-1 and tumor necrosis factor-
(TNF-
), and
activates vagal afferents. The aim of this study was to evaluate the
effect of peripheral LPS on rectal sensitivity and to specify the
mechanisms involved. Abdominal muscle contractions were recorded in
conscious rats equipped with intramuscular electrodes. Rectal
distension (RD) was performed at various times after LPS or
experimental treatments. In controls, RD significantly increased the
number of abdominal contractions from a threshold volume of distension
of 0.8 ml. At the lowest volume (0.4 ml), this number was increased
after administration of LPS (3, 9, and 12 h later), recombinant
human IL-1
(from 3 to 9 h), recombinant bovine TNF-
(from 6 to 9 h), and BrX-537A (from 6 to 12 h), a mast cell
degranulator. The effect of LPS was reduced by doxantrazole,
Lys-D-Pro-Thr, and soluble recombinant TNF receptor.
Vagotomy selectively amplified the response to LPS. We conclude that,
in vivo, intraperitoneal LPS lowers visceral pain threshold (allodynia)
through a mechanism involving mast cell degranulation and IL-1
and
TNF-
release and that the vagus nerve may exert a tonic protective
role against LPS-induced rectal allodynia.
endotoxins; rectal allodynia; mast cells; interleukin-1; tumor
necrosis factor-
; subdiaphragmatic vagotomy
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INTRODUCTION |
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THE GASTROINTESTINAL TRACT represents one of the body's largest interfaces with the outside environment. It possesses a complex immune system providing its defense against environmental threats including infection by viruses, bacteria, and parasites. Approximately one-third of patients with bacterial gastroenteritis develop chronic abdominal symptoms and signs of sensory changes in the gut (1). This entity, called "postinfectious irritable bowel syndrome" (PI-IBS), accounts for ~30% of all irritable bowel syndrome (IBS) patients. The major symptoms observed in patients with IBS include disordered colonic motility and acute or chronic abdominal pain. These patients exhibit an exaggerated sigmoid motor response to a variety of stimuli (41) and also have a lowered visceral sensory threshold to pain caused by balloon distension (35). Unfortunately, the pathophysiology of visceral hypersensitivity in patients with both IBS and bacterial infection is not precisely known.
Lipopolysaccharide (LPS), also known as endotoxin, is a cell wall
Gram-negative bacteria component. It induces a wide array of effects
after bacterial infection, including fever (18), sickness
behavior (17), and hyperalgesia. Indeed, several studies have shown that an intraperitoneal injection of LPS enhances pain responsiveness to various somatic stimuli (see Refs. 43 and 46).
Moreover, LPS-induced alterations in nociception depend on
proinflammatory cytokines released from monocytes and macrophages under
LPS stimulation such as interleukin (IL)-1, IL-6, and tumor necrosis
factor- (TNF-
). Indeed, an important role of inflammatory cytokines at the peripheral level has been recently recognized in
sensory hypersensitivity (44). For example, cutaneous
hyperalgesia can be produced by intraperitoneal injection of IL-1
(22) and TNF-
(42). Moreover, after
peripheral administration, LPS-, IL-1-
-, and TNF-
-induced
hyperalgesia requires vagal integrity because it is blocked by
subdiaphragmatic vagotomy (43, 44). This result agrees
with the observation that brain release of proinflammatory mediators,
including cytokines, is mediated in part by vagal afferents
(19), even though it does not appear to be the only
route for LPS/cytokine-to-brain communication (10).
Kanaan et al. (15) showed that intraplantar injection of
endotoxin produces local inflammation and delayed somatic hyperalgesia, mediated locally by IL-1, nerve growth factor (NGF), and
PGE2 (39). This hyperalgesic effect
starts 1-2 h after intraplantar endotoxin injection and peaks at
9 h in rats and 24 h in mice (16). Similarly, we
recently reported (3) that experimental mast cell
degranulation in vivo induces a delayed (6-12 h) increase in
sensitivity (allodynia) to rectal distension in awake rats. Indeed,
mast cells are involved in postinfectious (24) and
stress-induced (13) hyperalgesia, and their density is
altered in functional bowel disorders where, for example, an
accumulation of mast cells in the ileum has been demonstrated
(45). The anatomic arrangement of mast cells places them
in the first line of defense against injury or infection, particularly
for skin, airways, and gastrointestinal tract, sites that interface
directly with the external environment. These cells are well suited to
initiate an acute inflammatory process and, through interaction with
other tissue cells, to continue to maintain or modulate later response.
No studies have investigated the influence of systemic LPS on visceral
sensitivity, as was previously established for somatic sensitivity.
Consequently, the present study was designed to evaluate whether
intraperitoneal administration of endotoxin can initiate visceral
allodynia to rectal distension in rats and to determine the role of
peripheral IL-1 and TNF-
, the involvement of mast cells, and the
neuronal (vagus and/or other) pathway in LPS-related allodynia.
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MATERIALS AND METHODS |
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General Surgical Procedure
Animal preparation. Male Wistar rats (Harlan, Gannat, France), initially weighing between 200 and 250 g, were surgically prepared for electromyography according to a previously described technique (38). Rats were anesthetized by intraperitoneal injection of acepromazine (Calmivet, Vetoquinol, Lure, France) and ketamine (Imalgene 1000, Rhône-Mérieux, Lyon, France) at doses of 0.6 and 120 mg/kg, respectively. Three groups of three electrodes of NiCr wire (60-cm length and 80-mm diameter) were implanted bilaterally in the abdominal external oblique musculature just superior to the inguinal ligament. Electrodes were exteriorized on the back of the neck and protected by a glass tube attached to the skin. Animals were individually housed in polypropylene cages and kept in a temperature-controlled room (21°C). They were allowed free access to water and food (UAR pellets, Epinay, France). All protocols were approved by the Local Animal Care and Use Committee of Institut National de la Recherche Agronomique.
Electromyographic recording. Electromyographic recordings began five days after surgery. The electrical activity of abdominal striated muscles was recorded with an electroencephalograph machine (Mini VIII, Alvar, Paris, France) using a short time constant (0.03 s) to remove low-frequency signals (<3 Hz) and a paper speed of 3.6 cm/min.
Distension procedure. Rats were placed in plastic tunnels (6-cm diameter and 25-cm length) in which they could not move, escape, or turn around, to prevent damage to the balloon. Rats were exposed to this procedure over 3 days before rectal distension (RD) to minimize stress reactions during experiments. The balloon used for distension was an arterial embolectomy catheter (Fogarty, Edwards Laboratories, Santa Ana, CA). RD was performed by insertion of the balloon (2-mm diameter and 2-cm length) in the rectum, at 1 cm of the anus, the catheter being fixed at the tail with adhesive tape. The balloon was inflated progressively, in 0.4-ml steps, from 0 to 1.6 ml, each step of inflation lasting 5 min. To detect possible leakage, the volume of water introduced into the balloon was checked by complete removal with a syringe at the end of the distension period.
Temperature Recording
To measure body temperature (Tb), a thermistor probe (NTC type, code 10K3A1, Farnell, Villefranche sur Saône, France) was placed in the peritoneal cavity, using a previously described technique (26). Tb was recorded 5 days after surgery by connecting the thermistor probe to an electronic thermometer developed in our laboratory. It was calibrated to give an initial output of 0 V at 35°C with a sensitivity of 200 mV/°C. The temperature was monitored on a potentiometric recorder (L6514, Linseis, Selb, Germany) with a paper speed of 2 cm/h.Subdiaphragmatic Vagotomy
Surgery. Seven days before implantation of abdominal electrodes, abdominal vagotomies were performed as follows. Rats were anesthetized with ketamine and acepromazine (120 and 0.6 mg/kg ip, respectively). After midline laparotomy, the stomach and lower esophagus were visualized. The stomach was gently retracted beneath the diaphragm to clearly expose the ventral and dorsal trunks of the vagus nerve and covered with saline-moistened sterile gauze. Each vagal trunk was dissected from the esophagus and sectioned. The stomach was then returned to its normal position, and the incision was closed. Sham vagotomies consisted of the same operative procedure except that the vagal trunks were neither tied nor sectioned. Animals were returned to their home cages after the operation and were provided with food and water ad libitum. Normal food intake resumed within 3-5 days after vagotomy.
Verification procedure. The effectiveness of vagus nerve section was assessed 14 days after vagotomy by the sulfated cholecystokinin (CCK-8S) satiety test. Subdiaphragmatic vagotomy suppresses the blockade of food consumption induced by CCK-8S. Consequently, CCK-8S or saline was injected at the dose of 4 µg/kg ip after 20 h of food deprivation, and food intake was measured 1 h after injection.
Histological Mast Cell Counting Method
Intestinal tissue samples were put in Carnoy's solution immediately after the animals were killed, fixed for 24 h at room temperature, and then embedded in paraffin blocks using routine techniques. Sections were cut at a thickness of 5 mm and stained with hemalun-eosin for routine histological analysis or with Alcian blue-Safranin O for identification of intestinal mast cells. Three sections for each sample and each animal were analyzed by light microscopy, and the number of intact mast cells was counted for each section. For each animal, the number of intact mast cells per square millimeter of intestinal tissue was the mean of the values obtained for the three sections.Chemicals
LPS (from Escherichia coli, serotype 0111:B4; L3024, lot no. 38H4065) was purchased from Sigma-Aldrich (St. Quentin Fallavier, France) and was dissolved in saline (NaCl 0.9%) at a concentration of 1 mg/ml. BrX-537A (bromolasalocid ethanolate) was kindly supplied by Roche Laboratories (London, UK) and was dissolved in DMSO at a concentration of 2 mg/ml. Doxantrazole was obtained from Wellcome Research Laboratories (lot no. 59C72, Beckenham, UK) and was dissolved in DMSO (5 mg/ml). Recombinant human IL-1Experimental Protocol
Effect of LPS and role of mast cells.
These studies determined the effects of intraperitoneal injection of
LPS on rectal sensitivity and the involvement of mast cells using both
pharmacological and histological methods. In a first series of
experiments, two groups of rats were used. In the first group
(n = 6-8), rats were injected intraperitoneally with BrX-537A vehicle (DMSO) and, 48 h later, received BrX-537A (mast cell degranulator; 2 mg/kg ip). RDs were performed before (1 h,
control) and 3, 6, 9, 12, and 24 h after BrX-537A or vehicle administration. The dose of BrX-537A (2 mg/kg) has been found active in
a model of rectal sensitivity (3). Eight days later, the
same animals were injected intraperitoneally with LPS vehicle (saline)
and, 48 h later, received LPS (1 mg/kg ip). RDs were performed
before (
1 h, control) and 3, 6, 9, 12, and 24 h after LPS or
vehicle administration. The dose of LPS was chosen according to
preliminary experiments showing less reproducible and significant data
at lower doses (i.e., 0.1 and 0.5 mg/kg ip; Coelho et al., unpublished
observations). In the second group (n = 8), rats were injected, in a randomized order, with doxantrazole (5 mg/kg ip), a mast
cell stabilizing agent, or its vehicle 20 min before LPS or its
vehicle. RDs were performed before (
1 h, control) and 3, 6, 9, and
12 h after LPS administration. An 8-day interval was observed
between two single LPS administrations. The dose of doxantrazole (5 mg/kg ip) was chosen according to its efficacy in preventing mast cell
degranulation-induced rectal allodynia (3). The time
chosen between two single administrations of LPS (8 days) was judged to
be the minimum necessary time for a complete recovery from each LPS
treatment to eliminate a tolerance parameter of our LPS treatments.
Effect of LPS on Tb and behavior. Tb was recorded in a group of five rats. On day 1, animals were injected intraperitoneally with vehicle (saline, 1 ml/kg) after 1 h of temperature control recording. Temperature was monitored for a period of 24 h to establish a baseline. On day 2, the same animals were injected intraperitoneally with LPS (1 mg/kg) and Tb was recorded for 24 h. All measurements were performed at a subthermoneutral ambient temperature of 21.0 ± 1.0°C. All animals received saline or LPS between 8:30 and 9:00 AM.
Role of IL-1.
Two groups of rats were used to evaluate the role of IL-1
.
Group 1 (n = 9) was given rhIL-1
at a
dose (10 µg/kg ip) known to induce sickness behavior
(44). Vehicle was injected intraperitoneally for control
purposes. RDs were performed 3, 6, 9, and 12 h after rhIL-1
or
vehicle administration. Group 2 (n = 5)
received, in a randomized order, intraperitoneal injection of
tripeptide Lys-D-Pro-Thr (or vehicle) 30 min before LPS
injection, at the dose (10 mg/kg) known to antagonize IL-1
-induced
hyperalgesia (7) and to significantly reduce the
hyperalgesic effect of intraplantar LPS (39). The same
group received an injection of tripeptide alone. RDs were performed
before (
1 h, control) and 12 h after LPS (or vehicle) injection.
Eight days separated LPS/vehicle and LPS/tripeptide randomized treatments.
Role of TNF-.
Two groups of rats were used. Group 1 (n = 6) was injected intraperitoneally with rboTNF-
, or its vehicle, at a
dose (150 µg/kg) found to be active in a model of somatic
hyperalgesia (42). RDs were performed 3, 6, 9, and 12 h later. Group 2 (n = 6) was injected twice
intraperitoneally with sTNFR (total dose 2 mg/kg) or vehicle; the first
injection (1 mg/kg) was performed immediately before LPS (1 mg/kg ip)
or saline, and the second injection (1 mg/kg) was performed 90 min
later. RDs were performed before (
1 h, control) and 12 h after
LPS or saline injection. The delay of 90 min for the second injection
was chosen according to the efficacy of sTNFR, a procedure previously
validated in mice (27). Eight days separated LPS/vehicle
and LPS/sTNFR randomized treatments.
Effect of subdiaphragmatic vagotomy on LPS effect. Four groups of rats were used to determine the role of vagal nerves: sham vagotomy + vehicle (n = 5), sham vagotomy + LPS (n = 8), vagotomy + vehicle (n = 7), and vagotomy + LPS (n = 7). Rats were injected intraperitoneally with LPS (1 mg/kg) or its vehicle. RD was performed 12 h after vehicle or LPS injection.
Statistical Analysis
Statistical analysis of the number of abdominal contractions for each 5-min period during RD was performed by one-way ANOVA followed by Student's unpaired or paired t-test where relevant. Values are expressed as means ± SE. Tb values are presented as means ± SE and were compared by one-way ANOVA followed by Student's paired t-test. Mast cell numbers per square millimeter were analyzed using the Mann-Whitney U-test for unpaired data, and values are expressed as means ± SE. All differences were considered significant at P < 0.05. ![]() |
RESULTS |
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Effect of Intraperitoneal Injection of LPS on Rectal Sensitivity
Gradual RD increased the frequency of abdominal contractions in a distension volume-dependent manner. A volume of 0.8 ml was determined as the threshold at which RD induced a significant increase of the number of abdominal contractions compared with the predistension level (29). Saline-treated rats and untreated controls responded similarly to RD regardless of volume (0-1.6 ml) and time of distension (3, 6, 9, 12, and 24 h). LPS (1 mg/kg ip) increased the number of abdominal contractions for the 0.4-ml volume 3 (11.0 ± 1.8 contractions/5 min), 9 (10.5 ± 2.5 contractions/5 min), and 12 (16.1 ± 3.0 contractions/5 min) h after its administration compared with 3.4 ± 0.9 contractions/5 min for the control RD performed 1 h before LPS (Fig. 1). At other times (6 and 24 h) and other volumes (0.8-1.6 ml), abdominal responses were unaffected (P > 0.05) by LPS treatment (data not shown). On the basis of these data, we used the time of 12 h (maximal effect) to perform RDs in subsequent pharmacological investigations except for kinetic studies and determination of mast cell involvement.
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Effect of Intraperitoneal Injection of LPS on Tb and Behavior
The basal core temperature of rats was 37.8 ± 0.2°C. Intraperitoneal injection of vehicle (saline, 1 ml/kg) did not significantly (P > 0.05) modify the profile of Tb during the daytime period of recording. All rats displayed normal circadian changes in Tb, with lower daytime and higher nighttime Tb values (data not shown). LPS (1 mg/kg ip) significantly increased (P < 0.05) Tb between 1.5 and 9 h after injection. The Tb increase was characterized by the occurrence of two peak elevations, a first maximal Tb rise that peaked 2 h later (38.5 ± 0.2 vs. 37.5 ± 0.1°C of vehicle control) and a second maximal Tb rise that appeared 5.5 h later (38.4 ± 0.2 vs. 37.2 ± 0.1°C) (Fig. 2). Between 10 and 24 h, there were no differences in Tb between vehicle and LPS treatments. Concerning behavioral effects, rats injected with 1 mg/kg LPS showed a few signs of illness such as piloerection and lack of activity, but these did not last longer than 24 h. At the high dose used, no deaths were noted.
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Involvement of Mast Cells in LPS-Induced Rectal Hypersensitivity
As previously described (3), the number of abdominal contractions observed at the lowest volume of distension (0.4 ml) was significantly increased 6, 9, and 12 h after BrX-537A (2 mg/kg ip; 7.7 ± 2.0, 8.2 ± 1.8, and 13.1 ± 1.4 contractions/5 min, respectively, vs. 2.4 ± 0.5 for control RD) (Fig. 1). Doxantrazole (5 mg/kg ip) or its vehicle was given 20 min before LPS, and RD were performed 3, 6, 9, and 12 h after LPS. As in the previous series of experiments, animals receiving LPS after vehicle treatment showed an increase in the number of abdominal contractions only for the threshold volume of 0.4 ml at 3, 9, and 12 h after LPS (Fig. 3). Prior administration of doxantrazole (5 mg/kg ip) suppressed the abdominal response observed 3-12 h after LPS; compared with vehicle, doxantrazole significantly (P < 0.05) decreased the number of abdominal contractions at the 0.4-ml volume of distension, 3 (3.7 ± 1.7 vs. 11.0 ± 1.8 contractions/5 min for LPS + vehicle control) and 12 (5.7 ± 2.2 vs 16.1 ± 3.0) h after LPS administration (Fig. 3).
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Mast Cell Numbers After LPS Administration
To confirm the involvement of mast cells in LPS-induced visceral hypersensitivity and to compare the time course of mast cell degranulation with BrX-537A, histological examination of mucosal mast cells in the ileum and proximal colon was performed on rats treated with either BrX-537A or LPS. The number of intact mucosal mast cells in control animals was 215.1 ± 33.1 and 133.0 ± 17.9 cells/mm2 in the ileum and proximal colon, respectively. When rats were injected with BrX-537A (2 mg/kg ip), this number was significantly reduced (107.2 ± 17.6 and 81.2 ± 38.9 cells/mm2; P < 0.05) 1 h after BrK-537A administration. In contrast, rats given LPS presented a lower number of intact mucosal mast cells only 5 h (131.0 ± 22.3 and 72.8 ± 23.7 cells/mm2) after LPS administration (Table 1).
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Involvement of IL-1 in LPS-Induced
Delayed Visceral Allodynia
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Involvement of TNF- in LPS-Induced
Delayed Visceral Allodynia
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Involvement of Vagus Nerves in LPS-Induced Delayed Visceral Allodynia
CCK-8S significantly (P < 0.05) inhibited food intake in sham-vagotomized but not in vagotomized animals, thereby confirming the role of the vagus nerve in the CCK satiety test. Food intake was significantly decreased in CCK-injected sham-vagotomized rats compared with saline control sham-operated animals (1.8 ± 0.5 vs. 4.5 ± 0.4 g), whereas CCK-8S did not modify food intake in vagotomized animals compared with corresponding saline-injected vagotomized animals (3.7 ± 0.7 vs. 4.4 ± 0.4 g).After vehicle treatment, subdiaphragmatic truncal vagotomy did not
affect the number of abdominal contractions compared with sham-vagotomized animals at any volume of RD (Fig.
6). In sham-vagotomized animals, LPS
increased the number of abdominal contractions 12 h after its
administration at the RD volume of 0.4 ml (14.7 ± 1.4 vs.
4.9 ± 2.3 abdominal contractions/5 min for vehicle control), similarly to what was observed in intact animals. Surprisingly, marked
differences occurred between sham-vagotomized and vagotomized groups
after LPS injection (Fig. 6): the number of abdominal contractions at
the lowest volume of distension (0.4 ml) reached 23.9 ± 4.4 contractions/5 min in vagotomized animals, a value significantly higher
than that observed in sham-vagotomized animals (14.7 ± 1.4 contractions/5 min for sham + LPS value).
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DISCUSSION |
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The present experiments provide new insights regarding the effects
of LPS on visceral sensitivity as previously investigated for somatic
pain (43, 46). First, intraperitoneal injection of LPS
triggers a delayed lowering (9-12 h) of the threshold of rectal
distension-induced nociception in rats. Second, among the various
proinflammatory mediators released in response to LPS, IL-1 and
TNF-
appear to have a critical role in the genesis of LPS-induced
rectal allodynia. Third, the nociceptive response observed after LPS
administration is attenuated by doxantrazole, a mast cell stabilizer,
and histological studies confirmed gut mucosal mast cell degranulation
after LPS injection. Fourth, subdiaphragmatic vagotomy, surprisingly,
increases the magnitude of rectal allodynia induced by LPS. Together,
these data lead to the conclusion that intraperitoneal LPS evokes a
mast cell- and cytokine-dependent delayed rectal allodynia controlled
by vagus nerves.
In the first part of the study, we demonstrated that intraperitoneal LPS decreases rectal pain threshold but does not modify the magnitude of response to noxious volumes of distension. These data suggest that LPS released during infection favors an abnormal visceral sensitivity to mechanical stimuli, corresponding to a lowered threshold of barosensitivity, without affecting the visceral pain intensity evoked by noxious stimulation. The same observations were described previously after inflammation of colorectal mucosa by trinitrobenzene sulfonic acid in ethanol (29). The mechanisms evoking such abnormal visceral sensitivity are not yet fully understood. Such abnormal pain sensation, known as allodynia, could be related to a sensitization of low-threshold primary afferents, known to transmit nonpainful sensations in normal conditions and to be able to trigger postsynaptic nociceptive messages in inflammatory conditions. Such alterations have already been observed after nerve injury and cutaneous inflammation in somatic sensitivity (2, 12, 21). In the present study, the absence of change in the abdominal response for higher volumes may be related to the lack of sensitization of high-threshold afferents during the 12 h after LPS administration. Such a hypothesis might explain why LPS turns rectal perception into an abnormal one (allodynia) without modifying the pain response magnitude evoked by noxious stimuli.
We have also observed that intraperitoneal LPS enhances rectal sensitivity in two distinct periods, i.e., 3 h (early phase) and from 9 to 12 h (late phase) after its administration. We can attribute the early phase to a direct effect of endotoxin or pronociceptive mediators, such as PGE2 released from macrophages, acting on primary afferent terminals and the late phase to the subsequent development of inflammation with an intense activation of primary afferent fibers and changes in spinal or central processing (25, 32). In agreement with such a hypothesis, most reports related to endotoxin-induced somatic hyperalgesia have measured a decrease in the latency to cutaneous nociceptive stimulus that occurred in the first hour after intraperitoneal injection of LPS (44, 46). In addition, a model of localized inflammatory hyperalgesia was recently developed in rats using intraplantar injection of endotoxin in the hind paw (15). In this study, Kanaan et al. (15) also observed a peak of hyperalgesia 9 h after endotoxin injection with complete recovery 24 h later, and they explained this delayed response by a similar time-related occurrence of a localized inflammatory reaction.
Different patterns of body temperature profile (monophasic fever,
biphasic fever, and hypothermia/hyperthermia) have been described
depending on the dose of LPS used (36). At a high dose
(1 mg/kg), LPS triggers first a 1- to 3-h decrease of body temperature (hypothermia) followed by a long (6-24 h) period of fever (37, 47). In the present experiments,
intraperitoneal administration of a high dose of LPS, i.e., 1 mg/kg,
evokes a biphasic fever with two peaks of hyperthermia 2 and 5-6 h
later, but we have never observed an initial hypothermia in the first hour after injection. Such a discrepancy may be related to LPS preparation or serotype, rat strain, or route of administration, as
previously reported (11, 15).
In the third part of the study, we showed that LPS-induced delayed
allodynia is attenuated by an IL-1 receptor antagonist, the
tripeptide Lys-D-Pro-Thr. This result agrees with previous observations in which LPS-induced somatic hyperalgesia was also abolished by intraperitoneal administration of the IL-1 receptor antagonist (22) and by this tripeptide (39).
Moreover, Kanaan et al. (15) also suggested the
involvement of IL-1
in the mediation of both endotoxin-induced
thermal and mechanical hyperalgesia. Moreover, intraperitoneal IL-1
reproduces the nociceptive response of LPS on the pain threshold to
rectal distension with a time course of 3-9 h. Similarly, sTNFR,
which acts as a functional TNF antagonist, reduces LPS-induced delayed
allodynia (12 h), suggesting that TNF-
participates in the delayed
decrease of rectal pain threshold after LPS. These results are also in
agreement with data obtained for somatic pain. Indeed, the cutaneous
hyperalgesic effect of LPS can be blocked by TNF-
binding protein,
which acts as a functional TNF antagonist (42, 44).
Intraperitoneal TNF-
administration can also reproduce LPS
nociceptive response with a delayed maximal response appearing between
6 and 9 h. A long-lasting somatic hyperalgesia after intraplantar
TNF-
administration has also been reported (4).
Changes in visceral sensitivity related to LPS could be secondary to
the activation of chemosensitive nociceptors by inflammatory and/or
proalgesic mediators (32). Several chemicals produced by
local cells are capable of changing the sensitivity of nociceptors, including bradykinin, histamine, 5-hydroxytryptamine, neuropeptides such as substance P and calcitonin gene-related peptide, prostaglandins (5), and also cytokines (4, 7). LPS
stimulates the expression of a large number of cytokines, particularly
IL-1 and TNF-
, that may act directly on receptors found on
neurons or indirectly by stimulating the release of proalgesic
substances that can act on neurons in a cascadelike manner
(40). For example, IL-1, as well as LPS, stimulates the
arachidonic acid cascade, resulting in prostanoid production and
release (28) that sensitize primary afferent nociceptors
and augment the excitability of sensory afferents (5).
However, other mediators such as bradykinin can initiate both the
cascade of cytokine release that mediates hyperalgesic response to
endotoxins (9) and activation and sensitization of pain
nociceptors (14). In contrast to these data, IL-1 can also
induce hyperalgesia by acting directly on high-threshold mechanoreceptors, leading to a decrease in latency of neuronal discharges, to a lowering of the threshold, and to an increase of
neuronal response to mechanical and thermal stimulation
(10). In fact, these observations suggest a dual action of
IL-1
on different structures such as immune cells and the endings of
terminal neurons, depending on the pathophysiological context.
Concerning the potential involvement of TNF-
in the LPS cascade,
several lines of evidence suggest that TNF-
can exert its effects
through a cascade of cytokine release rather than by a direct
stimulation of sensory afferent neurons. Indeed, TNF-
is the first
cytokine released after LPS administration, and it reaches a maximal
plasma concentration after 1 h (48). In fact, TNF-
may produce hyperalgesia by inducing the secondary release of IL-1
,
because its effect can be blocked by an IL-1 receptor antagonist
(42). In carrageenan-evoked somatic hyperalgesia, the same
mechanism has been reported: bradykinin induces the release of TNF-
,
which in turn stimulates the release of other hyperalgesic cytokines
(IL-1
, IL-6, and IL-8) responsible for the generation of
cyclooxygenase products and sympathomimetic amines (8). In
consequence, these two cytokines seem to play a role in LPS-induced
visceral hypersensitivity at different levels and in a cascade.
Doxantrazole, a mast cell stabilizer, when injected before LPS, prevents both the early (3 h) and the late (12 h) phase of LPS-induced rectal allodynia. Consequently, mast cell activation appears to be involved in the cascade of reactions leading to the nociceptive response related to LPS administration. Concerning the early phase (3 h), a previous report showed that LPS can directly degranulate mast cells with production of cytokines without substantial release of preformed mediators by exocytosis (20). Moreover, other previous studies report the importance of various immune cells, particularly resident macrophages, from the liver in the production of somatic hyperalgesia appearing early within 1 h after intraperitoneal LPS administration (43, 44). Together, these observations permit us to suggest that the early phase could be linked to the activation of immune cells, and particularly mast cells, present in sites other than the gut wall because this early nociceptive response is abolished by previous treatment with a mast cell stabilizer, doxantrazole, and because no immediate mucosal mast cell degranulation is observed histologically in the gut. Concerning the late phase (12 h), we suggest that this phase is related to the development of an inflammatory reaction triggered by LPS and involving a delayed gut mucosal mast cell degranulation. Indeed, our histological study shows a decrease in mucosal mast cell number occurring 5 h after LPS administration, and this phase is abolished by doxantrazole. Moreover, this hypothesis is in agreement with a previous study showing that BrX-537A, a potent gut mucosal mast cell degranulator (30), promotes only a delayed rectal allodynia observed from 6 to 12 h after its administration (3). Furthermore, we have shown here that BrX-537A triggers an immediate (<1 h) mucosal mast cell degranulation in the gastrointestinal tract. Consequently, we can hypothesize that the late phase of rectal allodynia, consecutive to intraperitoneal LPS administration, is linked to resident mast cell degranulation localized in the intestinal tract.
Vagus nerve serves as an informational highway for inflammatory signals
from the periphery, and bilateral truncal vagotomy abolishes a wide
range of behavioral and neural effects of peripheral administration of
LPS. A part of the "illness" signals elicited by intraperitoneal
cytokines or LPS is relayed directly to brain primarily by the vagus
nerve, which activates a centrifugal pain facilitatory pathway. Indeed,
proinflammatory cytokines (IL-1 and TNF-
) produce somatic
hyperalgesia by activating vagal afferents (42, 44). In
contrast, our data demonstrate that total subdiaphragmatic vagotomy
amplifies LPS-induced rectal allodynia and thus that the vagus nerve
has a protective effect against LPS-induced visceral allodynia.
Chemical, electrical, or physiological activation of cardiopulmonary,
diaphragmatic, or subdiaphragmatic vagal afferents can result in either
facilitation or inhibition of nociception in some species, depending on
the intensity of stimulation (for review, see Ref. 31). For example,
high-intensity stimulation of vagal afferents activates spinal
descending inhibitory systems, whereas low intensity stimulates pain
facilitatory circuits (34). Therefore, it can be
postulated that, according to the intensity of the stimulus applied in
our study, vagal afferent fibers can participate in a feedback loop
directly controlling chemical nociceptive inputs from the periphery by
activating descending antinociceptive pathways. However, there is
substantial evidence that sensory neurons have a protective role
against injury to the gut (6, 33). Indeed, worsening of
inflammation has been observed in acute colitis models after perivagal
capsaicin pretreatment, suggesting a direct protective function of
vagal afferents on colonic mucosa against inflammation
(23). Thus we can hypothesize that similar processes may
underlie the protective action of vagus nerve against local
inflammatory reactions resulting from LPS administration. However, it
can also be hypothesized that the ability of total subdiaphragmatic
vagotomy to reduce LPS hyperalgesia, in some studies, or to amplify the
same response, as seen in our data, may be due to differences in the
dose of LPS applied. Indeed, the dose of 1 mg/kg is very high compared
with doses used in somatic models (16, 43), inducing, in
particular, fever and behavioral or locomotive disturbances. In
consequence, the vagus nerve appears to possess a powerful role in
mediating peripheral immune signals to the brain, but its role must be
different according to the nature and/or the amplitude of the
aggression and the pathophysiological context developed after LPS
infection. It is clear, however, that multiple circuits mediating pain
responses exist and are activated under different circumstances.
In summary, the present study indicates for the first time that
peripherally injected LPS lowers the visceral pain threshold to rectal
distension, this allodynia being observed between 9 and 12 h, and
that this effect is mediated by two cytokines, IL-1 and TNF-
, and
involves mast cell degranulation. Because intraperitoneal administration of LPS is associated with inflammatory reactions in the
gut, this result adds some insights into possible mechanisms by which
immune reactions of the brain-gut axis may participate in the genesis
of visceral allodynia.
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ACKNOWLEDGEMENTS |
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We thank C. Betoulieres, L. Ressayre, and I. Lorette for technical assistance and Institut National de la Recherche Agronomique and Solvay-Pharma Laboratories for financial support.
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FOOTNOTES |
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This work was presented in part at the 16th International Symposium on Gastrointestinal Motility, Lorne, Victoria, Australia, February 15-20, 1998 (3a).
Address for reprint requests and other correspondence: L. Buéno, INRA, NGN Unit, BP 3, F-31931 Toulouse, France (E-mail: lbueno{at}toulouse.inra.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 September 1999; accepted in final form 17 April 2000.
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