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Vol. 302, Issue 2, 659-665, August 2002
Departments of Cell Biology, Physiology, and Immunology (D.T., P.V.) and Pharmacology (R.T., D.d.M.), Universitat Autònoma de Barcelona, Bellaterra, Spain
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Nerve growth factor (NGF) could be involved in the development of hyperalgesia as well as in nervous remodeling consequence of inflammation. Both dysmotility and increase of visceral sensitivity have been described in functional gastrointestinal disorders such as irritable bowel syndrome. Trichinella spiralis-infected rats show an exacerbated spontaneous motility and a significant increase of the excitatory response to cholecystokinin (CCK), both associated with a reversible inflammatory process and the hypertrophy of the muscle layers. In this study we determined the intestinal expression of NGF mRNA by polymerase chain reaction and NGF by enzyme-linked immunosorbent assay. We implanted serosal strain gauge transducers on duodenum, jejunum, and ileum of anesthetized Sprague-Dawley rats to record circular muscle contractions. The experimental protocol included the evaluation of intestinal spontaneous motor activity (SMA), the response to CCK-8, and the ascending contraction induced by electrical mucosal stimulation. This protocol was performed in healthy and infected nontreated rats, in healthy rats with an NGF antibody treatment (1.6 mg/rat i.p.), and in infected rats with the same treatment applied at 0 or 3 days postinfection. NGF and NGF mRNA levels in the bowel were increased during inflammation. Although anti-NGF treatments did not prevent or reverse inflammatory response, the treatment was effective in preventing the motor alterations induced by the T. spiralis infection, i.e., inhibited increased SMA, reversed altered response to CCK, and reversed in part exacerbated response to electrical stimulation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Motor
alterations associated with clinical symptoms such as diarrhea,
constipation, and abdominal pain have been described in patients
affected by irritable bowel syndrome (IBS) (Harvey and Read, 1973
;
Azpiroz, 1999). Although IBS pathogeny is still undetermined, increased
sensitivity to gastrointestinal reflexes resulting from nerve
remodeling after a remote infection has been suggested as a possible
cause of these symptoms (Azpiroz, 1999; Camilleri, 2001). Both
intestinal hypermotility and nerve remodeling have been described in
several animal models using experimental parasite infection (Castro et
al., 1976; Palmer et al., 1984; Stead, 1992). In fact, the adaptive
response to intestinal parasites has been suggested as a paradigmatic
defense response of the intestine against external pathogens. For this
reason, experimental parasite infection has been widely used as model
to understand pathogenesis of intestinal functional disorders
(Blennerhassett et al., 1992; Stead, 1992; Hogaboam et al., 1996;
Torrents and Vergara, 2000).
Recently, we demonstrated that Trichinella spiralis-infected
rats show an increased spontaneous motor activity (SMA), increased ascending contraction of the peristaltic reflex, and an abnormal response to cholecystokinin (CCK) (Torrents and Vergara, 2000). The
control mechanisms involved in these responses indicated that both
intrinsic and extrinsic nervous systems of the bowel can be altered
during intestinal response to the parasite and become the cause of the
motor disturbances.
Nerve growth factor (NGF) is a protein of the family of neurotrophins
involved in the functionality of sensory nerves (Urschel et al., 1991),
and it has been implicated in the development of hyperalgesia during
inflammation (Lewin et al., 1994; Woolf et al., 1994; McMahon, 1996;
Theodosiou et al., 1999). The expression of both NGF and its receptors
by immune cells such as mast cells, lymphocytes, and basophils has been
described previously (Leon et al., 1994; Otten et al., 1994;
Levi-Montalcini et al., 1996; Sawada et al., 2000). Moreover, a
correlation between proliferation of vagal pathways and hyperplasia of
mast cells has been demonstrated in experimental parasite infection
(Stead, 1992).
Previous studies showed the presence of NGF in the intestine of adult
rats (Weskamp and Otten, 1987) and its production in vitro by
intestinal epithelial cells (Varilek et al., 1995). Moreover, a recent
study in a colitis model in rats showed a protective role of NGF
(Reinshagen et al., 2000). However, the involvement of NGF in the nerve
remodeling associated to intestinal hypermotility has not been studied yet.
The hypothesis of our study was that NGF might be involved in the genesis of hypermotility consequence of parasite infection and therefore in the exacerbation of intestinal reflexes observed in functional gastrointestinal disorders. Thus, the objectives of this study were to 1) determine the presence of NGF in the rat intestine and the possible overexpression of this neurotrophin during intestinal inflammation; 2) elucidate the possible role of NGF in the in vivo motor alterations described in T. spiralis-infected rats by means of a treatment with anti-NGF antibodies; 3) evaluate either the preventing or therapeutic value of this immunoneutralization using two different schedules for treatment, before and after inflammation was developed; and 4) evaluate by histopathology the severity of the inflammation.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals. Male Sprague-Dawley rats (Charles River, Lyon, France), 8 to 10 weeks old and weighing 300 to 350 g, were used in this study. They were kept under conventional conditions in a room with controlled temperature and photoperiod (12:12 h). Animals were specific pathogen free when purchased, and during the experimental period they were periodically checked for absence of intestinal parasites. Animal weight as well as food and water consumption were monitored daily.
T. spiralis Infection.
Rats
were infected by administering 1.0 ml of 0.9% saline solution
containing 7500 T. spiralis larvae by gavage. The
larvae were obtained from female CD1 mice infected 30 to 90 days before by a modification of the method described by Castro and Fairbairn (1969).
NGF mRNA Determination and NGF ELISA Assay.
For NGF mRNA and
protein determination, tissue samples from the jejunum of healthy rats
and of 3 day postinfection (PI)-infected rats were taken immediately
after killing the animals. In accordance with approval euthanasia
procedures, animals were first stunned and then killed by decapitation.
A 10-cm segment of the mid-jejunum was cut and divided into four equal
parts. Each sample was then weighted and stored immediately at
70°C.
) generating a fragment of 288 pb. As a
control of the efficiency of cDNA synthesis we amplified GAPDH (upper
primer 5'-ccgccccttccgctgatgcc-3' and lower primer 5'-atgagcccttccacgatgcc-3'), generating a fragment of 140 pb to test
the efficiency of cDNA synthesis. The polymerase chain reaction amplification protocol was as follows: 35 cycles at 95°C for 1 min,
55 or 50°C (NGF or GAPDH, respectively) for 1 min, and 72°C for 1 min; the final extension was for 5 min at 72°C. Amplified products
were electrophoresed on 2% agarose gel, stained with ethidium bromide,
and photographed under UV light. To have a semiquantitative measure of
mRNA NGF we calculated a ratio between band intensity of NGF and GAPDH
for each sample.
For NGF protein extraction the frozen samples were ground up into a
powder with a pestle and mortar, and then resuspended in extraction
buffer (1.5 g of intestine/ml of buffer) containing the protease
inhibitors phenylmethylsulfonic acid (0.1 mM), benzethonium chloride
(0.1 mM), aprotinin (7 µg/ml), and EDTA (4 mM) (all purchased from
Sigma-Aldrich, St. Louis, MO). The NGF ELISA assay (Roche Applied
Science, Manheim, Germany) was used according to the
manufacturer's instructions to determine the amount of NGF in rat
tissues. Briefly, anti (2.5 S, 7S)-NGF antibody (clone 27/21) was used
as a capture antibody at a concentration of 0.67 µg/ml. Unspecific
binding was prevented with 0.5% bovine serum albumin in binding buffer at 37°C for 30 min and washed eight times before adding samples and
standards. The plates were incubated overnight at 4°C and then washed
extensively. 
-Galactosidase-linked anti- (2.5 S, 7S)-NGF antibody
(clone 27/21) was applied (4 U/ml) and incubated for 4 h at
37°C. The color intensity was determined photometrically (570 nm)
after 30 min of incubation at 37°C with the substrate (clorophenol
red--D-galactopyranoside). For each plate a mouse NGF- standard curve was performed in parallel.
Animal Groups and NGF Treatments. Six groups of rats were studied: 1) nontreated healthy rats (cNT) (n = 6); 2) healthy rats treated with polyclonal neutralizing NGF (anti-NGF) antibody (Sigma-Aldrich) (cNGF) (n = 4); 3) nontreated infected rats (iNT) (n = 6); 4) infected rats treated with an unspecific IgG (Sigma-Aldrich) (iIgG) (n = 4); 5) infected rats treated with anti-NGF 1 h before infection (NGF0) (n = 4); and 6) infected rats treated with anti-NGF on day 3 PI (NGF3) (n = 6).
Both anti-NGF and IgG treatments consisted of a single intraperitoneal dose of 1.6 mg of the correspondent antibody in 1 ml of sterile 0.9% saline solution. These doses were calculated from data from a previous study using the same antibody (Reinshagen et al., 2000). Because both
animal experimental data (Watkins et al., 1997; Murthy et al., 1999)
and protocols with antibodies followed in humans (Targan, 2000) prove
that IgG remains in high concentrations in blood for prolonged periods,
we considered a single injection sufficient to maintain IgG
concentration for the duration of the experiment.
Animal Preparation for Motility Studies.
After a fasting
period of 12 to 16 h, animals were prepared for the experimental
procedures as described previously (Torrents and Vergara, 2000).
Briefly, anesthesia was induced by inhalation of halothane to allow
cannulation of the right jugular vein. Level III of anesthesia was
maintained for the rest of the experimental protocol with thiopental
sodium bolus infusion in the jugular vein as required. Body temperature
was maintained at 37°C by placing the animal on a heating pad. The
abdomen was opened and three strain-gauges 3 × 5 mm (Hugo Sachs
Elektronik, March-Hugstetten, Germany) were placed to record
circular muscle activity and sutured to the intestinal wall of the
duodenum, proximal jejunum, and ileum, respectively. Strain-gauges were
connected to high-gain amplifiers (MT8P; Lectromed Ltd, Letchworth,
Herts, UK), and amplified signals were sent to a recording unit
(PowerLab/800; ADInstruments Pty Ltd., Castle Hill, Australia)
connected to a PC running PowerLab software. Finally, two electrode
holders each with two platinum electrodes (WPI, Sarasota, FL) were
inserted into the intestinal lumen at 1 cm distally to the strain-gauge
of the duodenum and the ileum, respectively, to induce ascending
excitation of the peristaltic reflex by mucosal electrical stimulation
(EMS) as described previously (Giralt and Vergara, 2000). The Ethical
Committee of the Universitat Autònoma de Barcelona approved all
experimental procedures.
Evaluation of Motor Parameters.
After an equilibration
period of 20 min, spontaneous motor activity (SMA) was evaluated for
1 h. Then, CCK-8 (Peptide Institute, Inc., Osaka, Japan) (3 × 10
9 mol/kg/10 min) was intra-arterially
infused. After at least 1 h to allow the return to basal
conditions, mucosal electrical stimulation of the duodenum and ileum to
elicit ascending excitation was applied at 30 V, 0.6 ms and 2 and 6 Hz.
Each stimulus was applied for 30 s, and the polarity of the
stimulating electrodes was reversed at 15 s. Afterward, i.v.
administration of
N
-nitro-L-arginine
(Sigma-Aldrich) (L-NNA)
(105 mol/kg) was performed and the pattern of
electrical stimulation repeated 15 min later. Afterward, atropine
(Merck, Darmstadt, Germany) (0.3 mg/kg) was i.v. infused as a bolus and
5 min after the electrical stimuli were repeated. The two frequencies
of stimulus and the consecutive infusions of
L-NNA and atropine had the objective to evaluate
the functionality of inhibitory innervation and to differentiate the
excitatory response in two components (sensitive and resistant to atropine).
Capsaicin Studies.
To demonstrate implication of afferent
pathways on the altered intestinal response to CCK during inflammation,
we prepared a group of infected rats (n = 4) for
motility studies as we described above but also provided with two
intraluminal cannulas inserted through the same incision that the
intraduodenal electrode holder. These cannulas were used to instill
capsaicin into the intestinal lumen. One cannula was directed to the
duodenum and the other to the jejunum to ensure the complete diffusion
of the capsaicin in these two intestinal segments. The protocol we
followed with these animals was as follows: after 1 h to record
spontaneous motor activity, we administered a dose of CCK-8
intra-arterially (3 × 109 mol/kg/10 min)
(control response). Afterward, we infused capsaicin (Sigma-Aldrich) for
30 min (7.2 mg/kg in 2 ml of saline solution with 5% Tween 80 and 5%
dimethyl sulfoxide) and saline solution alone for 30 min more to wash
the intestinal lumen. Finally, we repeated CCK-8 infusion and compared
responses before and after capsaicin treatment. A sham-treated group
(n = 4) where only the solvents used to dissolve
capsaicin were instilled was included to identify any possible effect
of the solvents.
Data Analysis. The frequency of duodenal spontaneous motor activity in contractions per hour (c/h) was manually analyzed. The area under curve expressed in grams × seconds was measured for the whole period of electrical stimulation and CCK-8 infusion. All data were expressed as mean ± S.E.M, and statistical analysis of these parameters was performed using one-way analysis of variance and Bonferroni post-test. Moreover, ascending excitation after L-NNA and atropine were compared with their control response by a paired t test.
Histological Study. After finishing the experimental protocol, samples of jejunum were obtained, fixed for 48 h in 10% neutral buffered formalin, embedded in paraffin, cut into 5-µm sections, and stained with hematoxylin-eosin according to standard procedures. A scoring based on the inflammatory cell infiltration was used to evaluate the inflammatory process. At the same time, thickness of intestinal muscular layers was measured with a scored microscope. At least four different measures were taken from any sample, and samples from at least four animals of each group were used for the evaluation of the muscle thickness.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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NGF ELISA Assay and NGF mRNA Determination.
Both NGF protein
and mRNA levels were higher in the intestinal tissue from infected rats
than in healthy control rats (Fig. 1, A
and B, respectively). Samples from infected animals showed increased
levels of NGF (0.85 ± 0.26 pg of NGF/ml; n = 6)
compared with samples from healthy rats (0.09 ± 0.05 pg of
NGF/ml; n = 4). Intestinal samples from infected
animals also showed increased levels of NGF mRNA expression (1.088 ± 0.06; n = 6) compared with healthy rats (0.71 ± 0.13; n = 3).
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Intestine SMA.
Spontaneous activity was different in healthy
and infected rats. In infected rats, a clustered pattern substituted
the single contractions observed in the intestine of healthy rats. In
consequence, the analysis of contraction frequency showed a significant
difference (12.9 ± 3.4 c/h in cNT versus 29.3 ± 4.6 c/h in
iNT). This abnormal motility was reversed by early treatment with
anti-NGF (9 ± 3.5 c/h in NGF0) but not by the late treatment with
the anti-NGF (39.4 ± 7.6 c/h in NGF3) or with unspecific IgG
(30 ± 7.1 c/h in iIgG). Treatment of healthy rats with NGF
antibody did not cause any alteration on the SMA (14.3 ± 2.2 c/h
in cNGF). Statistical differences are shown in Table
1, and an example of these results is
shown in Fig. 2. After L-NNA
infusion during the experimental protocol an increase of spontaneous
motor activity was observed in all the groups without any significant
difference between them (data not shown).
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Response to CCK and Capsaicin Studies.
CCK induced an abnormal
response in the intestine of nontreated infected animals and in those
treated with the unspecific IgG. This response consisted of an
increased excitatory response in the duodenum concomitant with an
excitatory response of the jejunum. In contrast, both groups of animals
treated with the NGF antibody showed a response that was similar to
noninfected animals. This response is characterized by the excitation
of the duodenum while the jejunum remains quiescent (Fig.
3; Table 1).
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Response to EMS.
Duodenal response to EMS (Fig.
5) increased during intestinal
inflammation both at low frequency, 2 Hz, and at high frequency, 6 Hz.
Only the early treatment with anti-NGF (NGF0) reverted the exacerbated
response to 2 Hz.
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Histological Study.
Intensity of the inflammatory lesions and
thickness of circular muscle layer observed in the different groups is
summarized in Table 2. In cNT and in cNGF
groups no lesions were observed in the jejunal mucosa and submucosa. In
contrast, in iNT, and in animals infected and treated with NGF or IgG
unspecific, a severe mixed, but mainly mononuclear inflammatory cell
infiltrate, was present in the lamina propia, submucosa, and to a lower
extent in the smooth muscular layers of the jejunum. Moreover,
intestinal inflammation was accompanied by a smooth muscle hypertrophy
of the intestinal muscle layers. This hypertrophy was not reversed by
any of the treatments used in this study.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study demonstrates that NGF is overexpressed in the small intestine as a consequence of T. spiralis infection and also that this neurotrophin plays an important role in the development of motor alterations. Furthermore, because treatment with an antibody against NGF either prevented or attenuated most of the abnormal motor changes, this treatment may be a useful therapy for motor disorders caused by hypermotility.
As shown by ELISA, the infected intestine had a 4-fold increase of NGF
protein versus the control tissue. This was due to a higher NGF
production in the gut as revealed by the increased level of mRNA. This
higher amount of NGF in inflamed tissue has also been described in
other inflammatory models such as rheumatoid arthritis (Aloe et al.,
1992), pancreatitis (Toma et al., 2000), and experimental colitis
(Reinshagen et al., 2000); and has been associated with the increased
nervous sensitivity during inflammation (Woolf et al., 1994).
In our case NGF overexpression was already significant at 3 days PI.
However, preliminary data from intestine samples at 12 and 18 h
already showed a slight increase on NGF mRNA (data not shown). These
results indicate that NGF overexpression is initiated soon after
inflammation and probably causes nerve remodeling necessary to induce
hypermotility that is already very significant in this model around 6 days PI (Torrents and Vergara, 2000). Our treatment schedule was
planned to prevent NGF action (NGF0) or to block NGF when it was
already overexpressed (NGF3).
The intestinal motor hyperactivity induced by T. spiralis is a well known model of intestinal
hypersensitivity. Inflammation, as a consequence of parasites invading
the mucosa, starts a few hours after infection, and it is restricted to
duodenum and jejunum mucosa (Dick and Silver, 1980). However,
hypermotility, characterized by clustered contractions, appears around
day 6 PI and reaches its peak around day 11 PI, concomitant with
parasite expulsion. In addition, both hyperplasia and hypertrophy of
the muscular layers of the whole small intestine, including the ileum,
are significant around day 6 PI but last more than 72 days, far longer than the parasite expulsion and inflammation resolution (Torrents and
Vergara, 2000). We chose to perform the experiments at 10 to 12 days PI
when hypermotility was fully developed.
Anti-NGF preventive treatment (NGF0) completely blocked the development of spontaneous hypermotility, clearly indicating that NGF has a role in the development of abnormal motility. The fact that the NGF3 group presented a hypermotility similar to infected nontreated animals indicates that treatment was unable to revert completely motor changes once inflammatory mechanisms were activated. However, spontaneous motor activity is a complex event where both intrinsic and extrinsic pathways are involved. Thus, it is necessary to study other parameters that allow identification of the diverse mechanisms involved. For this reason, we also studied the response to CCK and to electrical stimulation.
We have already described in detail the mechanisms of the response to
CCK in the experimental model used in this study: The excitatory
response is mediated by mucosal afferent stimulation, whereas the
inhibitory response observed in the jejunum is due to stimulation of NO
release at the neuromuscular level (Giralt and Vergara, 1999). During
inflammation, CCK excitatory response in the duodenum increases and the
jejunum strongly contacts instead of being inhibited (Torrents and
Vergara, 2000). In the present study we demonstrate that abnormal
response to CCK is also mediated by capsaicin-sensitive afferent
fibers, indicating that the abnormal response to CCK is due to an
exacerbated response of afferent innervation.
Both anti-NGF treatments, either at time 0 (NGF0) or once inflammation
was established (NGF3), were effective in preventing CCK abnormal
response. This indicates that exacerbation of afferent response is
dependent of the overexpression of NGF. Furthermore, NGF antibodies
could be a possible treatment for those intestinal syndromes where
there is abnormal hypersensitivity, such as IBS where abnormal
responses to CCK have also been described previously (Harvey and Read,
1973). However, from our study we cannot know whether anti-NGF
treatment prevented either the proliferation of afferent fibers
described during intestinal inflammation (Sharkey, 1992; Shea-Donahue
et al., 1997) or the hyperplasia of other cell types, such as mast
cells, which also increase in numbers in this experimental model and
that have been suggested as a cause of afferent proliferation (Stead,
1992).
Intraluminal electrical stimulation elicits the ascending contraction
of the peristaltic reflex. In contrast to CCK response and to
spontaneous motility, this response can be blocked by intraluminal application of lidocaine but not by capsaicin (Giralt and Vergara, 2000). In consequence, electrical stimulation acts on either
capsaicin-insensitive afferents or, more likely, on a wide number of
neurons from the submucous plexus.
Ascending contraction is enhanced in infected animals. The response to
electrical stimulation has been extensively studied in several models
of intestinal disease both in vivo and in vitro conditions, and three
hypothesis have been raised to explain the hyper-response observed: 1)
an impairment of the inhibitory innervation (Hogaboam et al., 1996); 2)
the alteration of the acetylcholine (Ach)/substance P response (Collins
et al., 1989); and 3) the hypertrophy of muscle layers (Blennerhasset
et al., 1992).
In relation to whether there is an impairment of NO innervation, our
results are not conclusive. Infusion of L-NNA gave a similar increase of spontaneous motility in all experimental groups. However, at 2 Hz, a frequency of stimulus that recruits inhibitory innervation (Daniel and Kostolanska, 1989), L-NNA did not
increase ascending excitation, suggesting an impairment of NO. Anti-NGF preventive treatment (NGF0) brought back L-NNA effect,
indicating a restoration of NO response. In a previous study, also
using the same experimental model (Torrents and Vergara, 2000), we
reported that there was not a clear alteration of NO innervation that
could explain the hyper-response observed. However, from the present study we can conclude that if there is a certain functional impairment of NO innervation, it ameliorates after anti-NGF treatment.
In addition, the increased ascending contraction could also be a
consequence of the remodeling of excitatory innervation. The excitatory
intrinsic network of the intestine has been extensively studied. Both
Ach and substance P are colocalized in the same intrinsic motor neurons
(Furness et al., 1992), and changes in the Ach versus the substance P
component of the ascending contraction have been suggested after
T. spiralis infection (Torrents and Vergara,
2000). Moreover, an increase of substance P concomitant with a decrease
of acetylcholine has been reported (Collins et al., 1989; Swain et al.,
1992). In the present study, both anti-NGF treatments, but mainly the
preventive one, reverted the increased response after atropine,
allowing us to postulate that NGF is also responsible for the
remodeling of Ach/substance P neurons.
In contrast, none of the treatments prevented hypertrophy of the muscle. Hypertrophy could have contributed to the fact that none of the treatments was completely effective in reverting ascending contraction.
Similarly, none of the anti-NGF treatments were effective in preventing inflammation. This finding is interesting because it indicates that nerve remodeling is a specific action of NGF and not a secondary phenomenon of inflammation. It could also explain why in some hyper-responsive syndromes of the intestine score of inflammation is not well correlated with to the severity of the symptoms. Moreover, NGF expression could be a good target for treatment of the hyper-responsive intestine.
In conclusion, NGF overexpression regulates the nerve remodeling that modifies the sensitivity of intestinal motor reflexes. This remodeling affects afferent as well as motor innervation. The similarity between this experimental model of hypermotility and clinical intestinal motor syndromes, such as IBS, allows us to suggest anti-NGF strategies as a treatment for these diseases.
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Acknowledgments |
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We thank Dr. M. N. Prats for the histology evaluation, A. Acosta for skillful technical assistance, and A. C. Hudson for editorial revision of the manuscript.
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Footnotes |
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Accepted for publication April 9, 2002.
Received for publication February 20, 2002.
This work was supported by Dirección General de Investigación en Ciencia y Tecnología Grant PM98-0171 and Comissionat per a Universitats i Reserca, Generalitat de Catalunya, Grants 1999SGR-00272 and 2001SGR-00214. We are also grateful to Universitat Autònoma de Barcelona for the financial support of D. Torrents.
DOI: 10.1124/jpet.102.035287
Address correspondence to: Dr. Patri Vergara, Unitat de Fisiologia, Facultat de Veterinaria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. E-mail: patri.vergara{at}uab.es
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Abbreviations |
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IBS, irritable bowel syndrome;
SMA, spontaneous
motor activity;
CCK, cholecystokinin;
NGF, nerve growth factor;
ELISA, enzyme-linked immunosorbent assay;
PI, postinfection;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
cNT, nontreated healthy rats;
cNGF, healthy rats treated with polyclonal neutralizing NGF;
iNT, nontreated infected rats;
iIgG, infected rats treated with an
unspecific IgG;
NGF0, infected rats treated with anti-NGF 1 h
before infection;
NGF3, infected rats treated with anti-NGF on day 3 postinfection;
EMS, electrical mucosa stimulation;
L-NNA, N-nitro-L-arginine;
c/h, contractions per hour;
NO, nitric oxide;
Ach, acetylcholine.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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monoclonal antibody in dextran sulphate-induced mouse colitis.
Aliment Pharmacol Ther
13:
251-260[CrossRef][Medline]. therapy.
Can J Gastroenterol
14 (Suppl C):
13C-16C..
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