Primary sensory neurons: a common final pathway for
inflammation in experimental pancreatitis in rats
Jaimie D.
Nathan1,
Ruth Y.
Peng2,
Yu
Wang3,
Douglas C.
McVey3,5,
Steven R.
Vigna3,4,5, and
Rodger A.
Liddle3,5
Departments of 1 Surgery,
2 Pathology, 3 Medicine, and
4 Cell Biology, Duke University Medical Center,
Durham 27710; and 5 Veterans Affairs Medical Center,
Durham, North Carolina 27705
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ABSTRACT |
We hypothesized that neurogenic
inflammation is a common final pathway for parenchymal inflammation in
pancreatitis and evaluated the role of primary sensory neurons in
secretagogue-induced and obstructive pancreatitis. Neonatal rats
received either the primary sensory neuron-denervating agent capsaicin
(50 mg/kg sc) or vehicle. At 8 wk of age, pancreatitis was produced by
six hourly injections of caerulein (50 µg/kg ip) or by common
pancreaticobiliary duct ligation (CPBDL). The severity of pancreatitis
was assessed by serum amylase, pancreatic myeloperoxidase (MPO)
activity, histological grading, pancreatic plasma extravasation, and
wet-to-dry weight ratio. Caerulein significantly increased MPO activity
and wet-to-dry weight ratio, produced histological evidence of
edematous pancreatitis, induced plasma extravasation, and caused
hyperamylasemia. CPBDL increased MPO activity and produced histological
evidence of pancreatitis. Neonatal capsaicin administration
significantly reduced tissue MPO levels, histological severity scores,
and wet-to-dry weight ratio and abolished plasma extravasation. These
results demonstrate that primary sensory neurons play a significant
role in the inflammatory cascade in experimental pancreatitis and
appear to constitute a common final pathway for pancreatic parenchymal inflammation.
caerulein; capsaicin; common pancreaticobiliary duct ligation; neurogenic inflammation; substance P
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INTRODUCTION |
NEUROGENIC
INFLAMMATION REFERS to the local arteriolar vasodilatation,
increased vascular permeability, edema, and neutrophil infiltration
that result following stimulation of nociceptive primary sensory
neurons (13, 21). This inflammatory cascade is mediated by
peripheral release of chemical transmitters including the neuropeptide
substance P (SP) from nerve terminals of depolarized primary sensory
neurons. Subsequent binding of SP to its receptor, the neurokinin-1
receptor (NK-1R), on target cells, such as immune cells and vascular
endothelial cells (3, 10, 29), and involvement of other
neurotransmitters lead to manifestations of inflammation, including
tissue edema and neutrophil infiltration.
Several recent studies have suggested a role for neurogenic factors in
the inflammation of pancreatitis. Figini et al. (6) demonstrated that administration of SP to mice stimulates plasma extravasation from postcapillary venules in the pancreas, and this
effect is blocked by the administration of antagonists to the NK-1R. In
1998, Bhatia et al. (2) reported that genetic deletion of
the NK-1R in mice markedly reduced the severity of secretagogue-induced
pancreatitis and pancreatitis-associated lung injury. NK-1R knockout
mice have also been shown to have a reduced severity of diet-induced
hemorrhagic pancreatitis and a markedly improved survival in this model
(26). In a more recent study, the same group reported that
NK-1R antagonists blocked plasma extravasation in a model of
secretagogue-induced pancreatitis in rats (9). Although it
is clear that activation of the NK-1R plays a central role in the
neurogenic inflammatory process, additional evidence is required to
demonstrate the involvement of primary sensory neurons in this cascade
and to determine whether neurogenic inflammation is a common final
pathway in the development of pancreatitis.
Although primary sensory neurons are well known to contain SP, studies
have also localized SP to intrinsic enteric nerve plexuses. With the
use of colchicine treatment to block axonal transport of neuropeptide
granules, Su et al. (38) demonstrated the presence of
local SP-immunoreactive neuronal cell bodies within the pancreas of
rats. In addition, upper abdominal sympathectomy was shown to reduce
SP-containing nerve fibers in the pancreas, further supporting the
notion that there are dual intrinsic and extrinsic origins of
SP-immunoreactive neurons in rat pancreas. Thus the relative
significance of each of these pathways to neurogenic pancreatic
inflammation in experimental pancreatitis also requires elucidation.
The specificity of primary sensory neurons for the actions of
capsaicin, a sensory neuron excitotoxin, provides a useful model for
the evaluation of these questions. Neonatal capsaicin administration has been demonstrated to cause permanent loss of
capsaicin-sensitive primary sensory neurons as well as an
irreversible reduction in SP in regions normally innervated by
these neurons (17, 19, 32). Selective destruction of
primary sensory neurons by capsaicin attenuates the inflammatory
response induced by peripheral nerve stimulation or by injection of
noxious substances (7, 8). The technique of neonatal
capsaicin denervation has been used to demonstrate that primary sensory
neurons modulate the severity of joint injury in a rat model of
adjuvant-induced arthritis (24). In addition, with the use
of this method, other investigators have shown that primary sensory
neurons mediate toxin A-induced intestinal inflammation
(28), dermal inflammation caused by application of mustard
oil to the rat hind paw (17), and irritant-induced tracheal edema (18).
In the current study, we tested the hypotheses that primary sensory
neurons play a role in pancreatic inflammation in experimental pancreatitis and that primary sensory neurons are a common final pathway for neurogenic inflammation in pancreatitis. More
specifically, we examined whether primary sensory denervation
diminishes inflammation in rat models of secretagogue-induced
pancreatitis and obstructive pancreatitis. We demonstrate that neonatal
capsaicin administration causes primary sensory denervation and reduces
the severity of pancreatic inflammation in both models of pancreatitis,
suggesting that primary sensory neurons are a common final pathway for
inflammation in experimental pancreatitis.
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MATERIALS AND METHODS |
Animal protocol and experimental design.
All animal experiments were performed with approval of the Duke
University Institutional Animal Care and Use Committee. Male newborn
Sprague-Dawley rats were purchased from Charles River (Wilmington, MA)
and housed in climate-controlled rooms with a 12:12-h light-dark cycle.
On day (of life) 2, animals received either the
primary sensory denervating agent capsaicin (50 mg/kg sc)
(17) or vehicle [absolute ethanol/Tween 80/isotonic
saline (10:10:80, vol/vol/vol)]. Animals were weaned at ~4 wk of age and then fed standard laboratory chow until a 14- to 15-h overnight fast before the experiment. Rats were permitted water ad libitum throughout the experiment. When rats were 8 wk of age, primary sensory
denervation was confirmed by testing the eye-wipe response to assess
chemogenic pain perception (25). The number of protective forepaw eye wipes was counted in the first minute after application of
0.1% capsaicin to the cornea of the right eye. As additional confirmation of primary sensory denervation, we observed that rats that
received capsaicin neonatally lacked the normal withdrawal response to
tail pinch and had a reduced eye-wipe response to corneal application
of dilute acid (0.1 M HCl). It also should be noted that the pancreata
of rats that received capsaicin neonatally were grossly and
histologically indistinguishable from the pancreata of rats that
received vehicle neonatally. Rats were assigned to the following
experiments: caerulein-induced pancreatitis or obstructive pancreatitis.
Caerulein-induced pancreatitis.
The cholecystokinin analog caerulein was purchased from Bachem
California (Torrance, CA) and dissolved in 0.1 M NaHCO3
followed by dilution in isotonic saline. Caerulein was prepared the
morning of the experiment and stored on ice. Caerulein was administered as six hourly intraperitoneal injections at a supramaximal stimulating dose of 50 µg/kg per injection (22). Control rats
received six hourly intraperitoneal injections of isotonic saline. In
one group, 1 h after the last caerulein or vehicle injection,
animals were euthanized and mixed arteriovenous blood was collected by
decapitation for measurement of serum amylase concentration. The
pancreas was then quickly removed and divided for histological grading
and for measurement of tissue myeloperoxidase (MPO) activity. In a second group, Evans blue was used to quantify pancreatic plasma extravasation (35).
Obstructive pancreatitis.
Rats were anesthetized with xylazine (25 mg/kg im) and ketamine (50 mg/kg im), and the abdomen was entered via midline laparotomy. The
common pancreaticobiliary duct was doubly ligated adjacent to the
duodenal wall (33). Control rats underwent laparotomy with
double ligation of the bile duct at the hilum of the liver. Postoperatively, animals were permitted laboratory chow and water ad
libitum. On postoperative day 3, animals were euthanized and mixed arteriovenous blood was collected by decapitation for measurement of serum amylase concentration. The pancreas was then quickly removed
and divided for histological grading and for measurement of tissue MPO activity.
Serum amylase concentration.
Mixed arteriovenous blood was centrifuged for 10 min at 1,500 g. The serum amylase concentration was measured by using the procion yellow starch assay previously described (20). A
standard curve was prepared by using crude type VI-B
-amylase
(Sigma, St. Louis, MO), and serum amylase levels were expressed as
milligrams per milliliter.
MPO activity.
Portions of the harvested pancreata were immediately frozen at
80°C. We measured the tissue activity of MPO, an enzyme produced by
neutrophils and used as a marker of inflammation associated with
neutrophil infiltration, as previously described using the substrate
tetramethylbenzidine (39). Pancreatic tissue MPO
activity was expressed as units per milligram total protein.
Histological grading.
Portions of the pancreata were fixed overnight at room temperature in a
pH-neutral, phosphate-buffered, 10% formalin solution. The tissue was
then embedded in paraffin, sectioned at 5 µm, stained with
hematoxylin and eosin, and coded for examination by a blinded pathologist unaware of the experimental design. The pathologist graded
the severity of pancreatitis using modified scoring criteria previously
described (37). The results were expressed as a score of
0-3 for the histological parameters of edema and neutrophil infiltration. Tissue necrosis was graded on a scale of 0-14 and consisted of the sum of parenchymal necrosis (graded from 0 to 7) and
fat necrosis (graded from 0 to 7). Total histological score is the
combined scores of edema, neutrophil infiltration, and necrosis.
Plasma extravasation.
The Evans blue technique (35) was used to quantify
pancreatic plasma extravasation in caerulein-induced pancreatitis.
Evans blue is known to bind quantitatively to albumin and therefore remains intravascular until gaps form in the endothelial cell layer
allowing leakage into tissues. One hour after the last caerulein or
vehicle injection, rats were anesthetized with xylazine (25 mg/kg im)
and ketamine (50 mg/kg im). A right femoral vein cutdown was performed,
and Evans blue (30 mg/kg of a 3% solution in isotonic saline) was
injected intravenously. After 7 min, rats were transcardially perfused
with 70 ml PBS containing 100 U/ml heparin sodium, followed by 200 ml
4% paraformaldehyde in PBS. The pancreas was removed, rinsed in
saline, gently blotted, divided, and weighed. One-half of each pancreas
was dried in a vacuum oven for 48 h at 70°C and
25 mmHg and
reweighed for determination of a pancreas wet-to-dry weight ratio as a
measure of pancreatic edema. Evans blue was extracted from the second
half of each pancreas by incubation in 2 ml formamide at room
temperature for 48 h. To quantify Evans blue extracted, the
optical density of the formamide extract was measured at a wavelength
of 620 nm. Absorbance was compared with a standard curve, and plasma
extravasation was expressed as nanograms of Evans blue extracted per
milligram dry tissue weight.
Statistical analysis.
Results are expressed as means ± SE. Statistical methods included
the Student's t-test for eye-wipe response and one-way
ANOVA with the Tukey's posttest for pancreatitis severity parameters (GraphPad Prism version 2.00, GraphPad Software, San Diego, CA). Statistical significance was set at P < 0.05.
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RESULTS |
Eye-wipe response.
To confirm primary sensory denervation, we assessed chemogenic pain
perception when rats were 8 wk old by evaluating the protective eye-wipe response to application of capsaicin to the cornea. In vehicle-treated rats, the mean number of ipsilateral forepaw eye wipes
following application of 0.1% capsaicin to the right cornea was ~40
(Fig. 1). In rats that received capsaicin
as neonates, this response was significantly reduced to only nine eye
wipes per minute (P < 0.0001; n = 28),
confirming primary sensory denervation.

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Fig. 1.
The eye-wipe response to corneal application of 0.1%
capsaicin. Neonatal capsaicin administration significantly reduced the
eye-wipe response to corneal application of capsaicin, confirming
primary sensory denervation. Results are expressed as means ± SE
(n = 28). *P < 0.0001 vs. vehicle.
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Caerulein-induced pancreatitis.
Administration of supramaximal stimulating doses of caerulein (6 hourly
injections of 50 µg · kg
1 · dose
1) to
noncapsaicinized adult rats produced evidence of pancreatitis, as
determined by serum amylase levels, tissue MPO activity, and histological grading. The serum amylase concentration in rats that
received caerulein increased 20-fold compared with vehicle-treated animals (P < 0.001; n = 6; Fig.
2). In addition, caerulein treatment caused a significant increase in the level of pancreatic MPO activity, from 0.015 ± 0.002 to 0.654 ± 0.228 U/mg (P < 0.01; n = 6; Fig. 3).
Histologically, caerulein treatment produced moderately severe pancreatitis characterized by pancreatic edema, neutrophil
infiltration, and parenchymal necrosis as well as an injury pattern of
acinar cell vacuolization (Fig. 4). All
histological severity parameters were significantly elevated in
noncapsaicinized rats receiving caerulein (Table
1), with an increase in total
histological score from 0.25 ± 0.11 to 6.08 ± 0.54 (P < 0.001; n = 6; Fig.
5).

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Fig. 2.
The effects of caerulein and neonatal capsaicin
administration on serum amylase concentration. Caerulein administration
significantly increased the serum amylase concentration, but neonatal
capsaicin administration had no effect on this caerulein-induced
elevation. Results are expressed as means ± SE (n = 6). *P < 0.001 vs. vehicle.
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Fig. 3.
The effects of caerulein and neonatal capsaicin
administration on pancreatic myeloperoxidase (MPO) activity. Caerulein
administration increased pancreatic MPO activity, and this effect was
significantly inhibited by neonatal capsaicin administration. Results
are expressed as means ± SE (n = 6).
*P < 0.01 vs. vehicle + vehicle;
P < 0.05 vs. vehicle + caerulein.
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Fig. 4.
The effects of caerulein and
neonatal capsaicin administration on pancreatic histoarchitecture.
Representative histological sections of rat pancreas fixed in 10%
neutral-buffered formalin, paraffin embedded, and stained with
hematoxylin and eosin from control (A), caerulein alone
(B), and caerulein + neonatal capsaicin
(C)-treated rats. Caerulein administration caused pancreatic
edema, neutrophil infiltration, and parenchymal injury and necrosis.
Neonatal capsaicin administration inhibited the effects of caerulein on
pancreatic histoarchitecture. Magnification: ×250.
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Fig. 5.
The effects of caerulein and neonatal capsaicin
administration on total histological score of pancreatitis. Caerulein
administration increased the total histological severity score of
pancreatitis, and this effect was significantly inhibited by neonatal
capsaicin administration. Results are expressed as means ± SE
(n = 6). *P < 0.001 vs. vehicle + vehicle; P < 0.001 vs. vehicle + caerulein.
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Neonatal capsaicin administration resulted in a reduction in the
caerulein-induced elevation in pancreatic MPO activity. Compared with
noncapsaicinized animals, capsaicinized rats receiving caerulein exhibited an 82% reduction in MPO activity (p < 0.05;
Fig. 3) to a level indistinguishable from vehicle-treated rats. In
contrast to noncapsaicinized rats, the capsaicin-treated animals
receiving caerulein developed markedly less pancreatic edema,
neutrophil infiltration, and parenchymal necrosis as illustrated
histologically (Fig. 4). As shown in Table 1, neonatal capsaicin
administration significantly reduced the scores of edema by 36%
(P < 0.01), neutrophil infiltration by 41%
(P < 0.01), and necrosis by 59% (P < 0.05). The total histological severity score was diminished by 47% in rats that were treated with capsaicin neonatally (P < 0.001; Fig. 5). Although the serum amylase concentration tended to be
lower in capsaicinized rats, there was no statistically significant difference compared with noncapsaicinized animals.
In a second group of rats, we used the Evans blue technique to quantify
plasma extravasation into the pancreatic interstitium in
caerulein-induced pancreatitis. Caerulein treatment of noncapsaicinized rats increased Evans blue extravasation by greater than fivefold, from
7.2 ± 3.3 to 37.8 ± 4.8 ng/mg dry weight (P < 0.001; n = 4; Fig. 6).
In addition, caerulein treatment caused a significant increase in the
pancreas wet-to-dry weight ratio, from 2.6 ± 0.3 to 7.2 ± 1.4 (P < 0.05; n = 4; Fig.
7). Compared with noncapsaicinized animals, capsaicinized rats receiving caerulein had an 85% reduction in Evans blue extravasation (P < 0.001; Fig. 6) to a
level indistinguishable from vehicle-treated rats. Similarly, neonatal
capsaicin administration diminished the caerulein-induced elevation in
pancreas wet-to-dry weight ratio (P < 0.05; Fig. 7) to
the level of vehicle-treated rats.

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Fig. 6.
The effects of caerulein and neonatal capsaicin
administration on pancreatic plasma extravasation, as measured by Evans
blue (EB). Caerulein administration induced EB extravasation in the
pancreas, and this effect was significantly inhibited by neonatal
capsaicin administration. Results are expressed as means ± SE
(n = 4). *P < 0.001 vs. vehicle + vehicle; P < 0.001 vs. vehicle + caerulein.
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Fig. 7.
The effects of caerulein and neonatal capsaicin
administration on pancreas wet-to-dry weight ratio as a measure of
tissue edema. Caerulein administration increased the pancreas
wet-to-dry weight ratio, and this effect was significantly inhibited by
neonatal capsaicin administration. Results are expressed as means ± SE (n = 4). *P < 0.05 vs.
vehicle + vehicle; P < 0.05 vs.
vehicle + caerulein.
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Obstructive pancreatitis.
Common pancreaticobiliary duct ligation (CPBDL) in noncapsaicinized
adult rats resulted in biochemical and microscopic evidence of acute
pancreatitis. Ligation of the common pancreaticobiliary duct caused a
significant increase in the level of pancreatic MPO activity, from
3.18 ± 1.04 to 10.59 ± 0.71 U/mg (P < 0.01; n = 4; Fig. 8).
Histologically, CPBDL produced severe pancreatitis characterized by
edema formation, neutrophil infiltration, acinar cell vacuolization,
and parenchymal and fat necrosis (Fig.
9). Both the edema score and the necrosis
score were significantly elevated in noncapsaicinized rats that
underwent ligation of the pancreaticobiliary duct (Table
2). Compared with rats undergoing biliary
duct ligation, the total histological score in the CPBDL group
increased from 1.38 ± 0.43 to 13.88 ± 1.20 (P < 0.001; n = 4; Fig.
10). The serum amylase concentration in
noncapsaicinized rats that underwent CPBDL did not statistically differ
from the control group (Fig. 11).

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Fig. 8.
The effects of duct ligation and neonatal capsaicin
administration on pancreatic MPO activity. Common pancreaticobiliary
duct ligation (CPBDL) increased pancreatic MPO activity, and this
effect was significantly inhibited by neonatal capsaicin
administration. Results are expressed as means ± SE
(n = 4). *P < 0.01 vs. vehicle + biliary duct ligation (BDL); P < 0.01 vs. vehicle + CPBDL.
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Fig. 9.
The effects of duct ligation and
neonatal capsaicin administration on pancreatic histoarchitecture.
Representative histological sections of pancreas fixed in 10%
neutral-buffered formalin, paraffin embedded, and stained with
hematoxylin and eosin from rats that underwent BDL (A),
CPBDL (B), and CPBDL + neonatal capsaicin
administration (C). CPBDL caused pancreatic edema,
neutrophil infiltration, and parenchymal injury and necrosis. Neonatal
capsaicin administration inhibited the effects of CPBDL on pancreatic
histoarchitecture. Magnification: ×250.
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Fig. 10.
The effects of duct ligation and neonatal capsaicin
administration on total histological score of pancreatitis. CPBDL
increased the total histological severity score of pancreatitis, and
this effect was significantly inhibited by neonatal capsaicin
administration. Results are expressed as means ± SE
(n = 4). *P < 0.001 vs. vehicle + BDL; P < 0.05 vs. vehicle + CPBDL.
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Fig. 11.
The effects of duct ligation and neonatal capsaicin
administration on serum amylase concentration. Serum amylase
concentrations among groups were not significantly different. Results
are expressed as means ± SE (n = 4).
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Neonatal capsaicin administration resulted in a reduction in the
CPBDL-induced elevation in pancreatic MPO activity. Capsaicinized rats
undergoing CPBDL had a 59% reduction in MPO activity
(P < 0.01; Fig. 8) compared with noncapsaicinized
animals. In contrast to noncapsaicinized rats, the capsaicin-treated
animals that underwent CPBDL demonstrated less pancreatic edema,
neutrophil infiltration, and parenchymal and fat necrosis
histologically (Fig. 9), although only the necrosis score was
significantly reduced, from 9.50 ± 1.23 to 4.88 ± 0.55 (P < 0.05; Table 2). The total histological severity
score was diminished by 43% in rats that were treated with capsaicin
neonatally (P < 0.05; Fig. 10). Serum amylase
concentrations were not significantly different in capsaicinized animals.
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DISCUSSION |
Recent studies have suggested that neurogenic factors play a
significant role in the inflammatory cascade in experimental pancreatitis. SP-induced pancreatic plasma extravasation in mice has
been demonstrated to be blocked by NK-1R antagonists (6). NK-1R knockout mice have been reported to have a markedly reduced severity of secretagogue-induced pancreatitis (2) and
diet-induced hemorrhagic pancreatitis (26). In addition,
plasma extravasation in a rat model of secretagogue-induced
pancreatitis is blocked by administration of antagonists to the NK-1R
(9). These studies have suggested an important role for SP
stimulation of the NK-1R in the pathogenesis of experimental
pancreatitis. However, the specific involvement of SP-containing
primary sensory neurons has not yet been reported.
It is well established that a subpopulation of cell bodies of primary
sensory neurons synthesize the undecapeptide neurotransmitter SP, which
is released from nerve endings following nerve stimulation and
depolarization (11, 12, 34). Furthermore, Won et al. (43) demonstrated by retrograde tracing and
immunohistochemistry that the rat pancreas is innervated by
SP-immunoreactive nerve fibers originating from dorsal root ganglion
cells. However, other studies (38) reported the presence
of SP-immunoreactive cell bodies in the pancreas after colchicine
treatment as well as partial abolition of SP-immunoreactive nerve
fibers by surgical denervation. These findings suggest that SP nerve
fibers in the pancreas originate from both intrinsic (i.e., enteric)
and extrinsic (i.e., primary sensory) cells. The potential contribution
of each of these sources of SP to neurogenic pancreatic inflammation in
pancreatitis is unknown. In this study, we hypothesized that primary
sensory neurons play a role in pancreatic inflammation in experimental
pancreatitis and that primary sensory neurons are a common final
pathway for neurogenic inflammation in pancreatitis. More specifically,
we examined whether primary sensory denervation would ameliorate pancreatic inflammation in rat models of secretagogue-induced and
obstructive pancreatitis.
We used the neonatal capsaicin administration model to produce primary
sensory denervation. Capsaicin, a chemical derivative of vanillyl amide
and the pungent agent in red pepper, has been used extensively as a
probe for primary sensory neuron mechanisms, and its value as a
pharmacological tool is dependent on the specificity of its neurotoxic
actions for primary sensory neurons. Systemic administration of a
single dose of 50 mg/kg capsaicin in neonatal rats causes permanent
degeneration of 90-95% of unmyelinated primary sensory neurons,
with no significant change in myelinated afferent fibers, and
irreversible depletion of sensory neuron SP (4, 23, 31,
32). Of particular importance is the finding in the
gastrointestinal tract that neonatal capsaicin administration ablates
primary sensory neurons while leaving intrinsic SP-containing neurons
intact (16). Investigators have used this denervation model to demonstrate the involvement of primary sensory neurons in
various inflammatory conditions, including toxin A-induced intestinal
inflammation (28), irritant-induced tracheal edema (18), and adjuvant-induced experimental arthritis
(24).
To confirm capsaicin-induced primary sensory denervation, we evaluated
chemogenic pain perception by counting the number of protective forepaw
eye-wiping movements in response to instillation of 0.1% capsaicin
into the eye (25). The eye-wipe response is a
well-accepted method of confirming primary sensory denervation, because
it provides gross behavioral evidence of impairment of chemonociceptive
primary sensory innervation. Furthermore, suppression of
chemonociceptive pain responses by neonatal capsaicin administration has been demonstrated to correlate with the prevention of neurogenic inflammatory responses (17). In our study, the eye-wipe
response was reduced by 78% in rats that received capsaicin as
neonates, and there was an increased latency of ~5 s in the
initiation of protective eye wipes after the instillation of capsaicin
into the eye (data not shown). Although these data suggest that primary sensory denervation was not complete, which is in keeping with prior
studies demonstrating degeneration of up to 95% of unmyelinated sensory afferent neurons, it is clear that neonatal capsaicin administration resulted in permanent loss of the majority of primary sensory neurons.
The current study demonstrates that repeated caerulein
administration in rats causes biochemical and histological evidence of
acute pancreatitis. Moreover, neonatal capsaicin administration causes
primary sensory denervation and reduces the severity of secretagogue-induced pancreatitis. To determine whether neurogenic inflammation is a common final pathway for parenchymal inflammation in
pancreatitis, we evaluated the role of primary sensory neurons in a
surgical model of obstructive pancreatitis. We report that ligation of
the common pancreaticobiliary duct in rats causes biochemical and
histological evidence of acute pancreatitis and that primary sensory
denervation induced by neonatal capsaicin treatment reduces the
severity of obstructive pancreatitis. These findings suggest that
primary sensory neurons play a critical role in the tissue inflammatory
response to injury in pancreatitis and that neurogenic factors
constitute a common final pathway for parenchymal inflammation in
pancreatitis. Furthermore, we propose that initial parenchymal injury,
by caerulein hyperstimulation or by common pancreaticobiliary duct
obstruction, generates a signal that activates primary sensory neurons
resulting in release of peptide transmitters, including SP, with
subsequent amplification and propagation of the inflammatory
cascade. Whether primary sensory neurons play a role in other models of
experimental pancreatitis, such as diet-induced hemorrhagic
pancreatitis and trypsin/taurocholate-induced pancreatitis, remains to
be determined.
It is worth noting that there were differences in the extent of effects
of capsaicin in the two models of pancreatitis. Specifically, the
percentage reductions in necrosis scores were 59% in caerulein-induced pancreatitis and 49% in obstructive pancreatitis; in total
histological score, 47% and 43%, respectively; and in tissue MPO
activity, 82% and 59%, respectively. It also should be noted that the
overall severity of pancreatitis differed between the two models, with obstructive pancreatitis being more severe. However, a difference in
severity was not unexpected, because the pathogenesis of
caerulein-induced pancreatitis differs from that of obstructive
pancreatitis. Because of the disparity in severity between the two
models, it is also not surprising that intervention (i.e., neonatal
capsaicin administration) had a slightly different effect in the two
models. It is possible that the effects of capsaicin were less
impressive in obstructive pancreatitis, because the severity of
pancreatitis in this model was greater than in caerulein-induced pancreatitis.
Plasma extravasation is an early and major component of neurogenic
inflammation in the trachea (1), urinary bladder
(30), and skin (36). In this study, using the
Evans blue technique, we have demonstrated that caerulein treatment in
rats stimulates pancreatic microvascular permeability and plasma
extravasation with edema formation. In contrast to the partial
reductions in biochemical and histological parameters of pancreatitis,
primary sensory denervation with neonatal capsaicin administration
completely abolished caerulein-induced pancreatic plasma extravasation
and edema. This finding is in agreement with earlier studies that reported that SP-induced pancreatic plasma extravasation in mice (6) and caerulein-induced pancreatic plasma extravasation
in rats (9) are abolished by antagonists to the NK-1R.
This suggests that secretagogue-induced plasma extravasation in rat
pancreas is mediated entirely by activation of primary sensory neurons, whereas other components of neurogenic inflammation, such as neutrophil infiltration and parenchymal necrosis, are predominantly, but not
entirely, mediated by sensory afferent neurons.
Neonatal capsaicin administration reduced the severity of tissue damage
induced by caerulein by significantly diminishing pancreatic edema,
neutrophil infiltration, parenchymal necrosis, and plasma
extravasation. Although serum amylase levels tended to be lower in the
rats that were treated with capsaicin neonatally, these data were not
significantly different. This finding suggests that
caerulein-stimulated amylase release is not mediated by primary sensory
neurons and does not correlate with the severity of experimental pancreatitis. In our model of obstructive pancreatitis, serum amylase
concentrations on postoperative day 3 after CPBDL did not
statistically differ from amylase levels in control animals. These data
are in agreement with Ohshio et al. (33), who reported that the elevated serum amylase concentrations resulting within 3 h of CPBDL in rats fell to the level of control rats by 6 h postoperatively. It is clear from our results, as well as from reports
of our colleagues, that serum amylase levels are variable throughout
the course of the disease and are not predictive of severity of
pancreatic inflammation.
Studies from another laboratory (40-42) using an
adult model of capsaicin administration to cause ablation of sensory
neurons have suggested a protective role of primary sensory neurons
and, specifically, calcitonin gene-related peptide in caerulein-induced pancreatitis. This technique to cause sensory denervation differs substantially from administration of capsaicin in the neonatal period.
Although adult capsaicin administration can ablate sensory neurons, the
effect of capsaicin in adult rats is not permanent, and sensory
deficits and neuropeptide depletion are often reversible (4). In addition, it is known that the neurotoxic effects
of capsaicin in adult rats are not as widespread as they are in
neonates, and thus the depletive effect on sensory neuropeptides is not as extensive (4, 15). Because of the confounding factors associated with adult capsaicin administration, we suggest that the
differences in sensory nerve ablation technique preclude an adequate
comparison between our results and the results of these prior studies.
In conclusion, the results of this study demonstrate that
caerulein-induced and obstructive pancreatitis in rats are mediated, in
part, by primary sensory neurons. Because neonatal capsaicin administration specifically destroys small, unmyelinated primary sensory neurons, the integrity of these afferent fibers is critical to
the development of pancreatic inflammation. It is important to note
that primary sensory neurons are known to contain several neuropeptides, including SP, calcitonin gene-related peptide, and
neurokinin A (5, 14, 27). Although SP is by far the most
potent inflammatory neuropeptide derived from primary sensory neurons,
calcitonin gene-related peptide has been demonstrated to induce
vasodilatation, and neurokinin A is a mediator of plasma extravasation
(14). Thus additional studies are required to elucidate
the role of each these neuropeptides as it relates to the reduction in
pancreatic inflammation following primary sensory denervation.
Nevertheless, the current study demonstrates the specific involvement
of primary sensory neurons in the inflammatory cascade of experimental
pancreatitis and the role of primary sensory neurons as a common final
pathway in pancreatic inflammation.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-38626, DK-50265, and DK-10106
and by a Dept. of Veterans Affairs merit review grant.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
R. A. Liddle, Dept. of Medicine, Box 3913, Duke Univ.
Medical Center, Durham, NC 27710 (E-mail:
liddl001{at}mc.duke.edu).
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.
June 5, 2002;10.1152/ajpgi.00105.2002
Received 18 March 2002; accepted in final form 30 May 2002.
 |
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