Group Title: Molecular Pain 2009, 5:54
Title: NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs) during inflammation induced visceral hypersensitivity
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Title: NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs) during inflammation induced visceral hypersensitivity
Series Title: Molecular Pain 2009, 5:54
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Creator: Suckow SK
Caudle RM
Publication Date: 40078
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Molecular Pain BioMedCentra


NMDA receptor subunit expression and PAR2 receptor activation
in colospinal afferent neurons (CANs) during inflammation induced
visceral hypersensitivity
Shelby K Suckowtl and Robert M Caudle*tl,2

Address: 'Department of Neuroscience, University of Florida College of Medicine, Gainesville, FL 32610, USA and 2Department of Oral and
Maxillofacial Surgery and Diagnostic Sciences, University of Florida College of Dentistry, Gainesville, Florida 32610, USA
Email: Shelby K Suckow; Robert M Caudle*
* Corresponding author tEqual contributors

Published: 22 September 2009 Received: 14 May 2009
Molecular Pain 2009, 5:54 doi:10.1 186/1744-8069-5-54 Accepted: 22 September 2009
This article is available from:
2009 Suckow and Caudle; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Visceral hypersensitivity is a clinical observation made when diagnosing patients with
functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be
attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons,
colospinal afferent neurons (CANs), co-localize with the NRI and NR2D subunits of the NMDA
receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and
PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation.
Recently, work has suggested that dorsal root ganglion (DRG) neurons expressing the transient
receptor potential vanilloid-l (TRPVI) receptor mediate inflammation induced visceral
hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG
neurons expressing the TRPVI receptor were lesioned with resiniferatoxin (RTX) prior to
inflammation and behavioral testing.
Results: CANs do not express the TRPVI receptor; therefore, they survive following RTX
injection. RTX treatment resulted in a significant decrease in TRPVI expressing neurons in the
colon and immunohistochemical analysis revealed no change in peptide or receptor expression in
CANs following RTX lesioning as compared to control data. Behavioral studies determined that
both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as
compared to controls. Immunohistochemical analysis demonstrated that the NRI cassettes, NI
and C of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore,
inflammation resulted in the activation of the PAR2 receptors expressed on CANs.
Conclusion: Our data show that inflammation causes an up-regulation of the NMDA receptor and
the activation of the PAR2 receptor expressed on CANs. These changes are associated with a
decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned
animals. Therefore, these data suggest that CANs contribute to visceral hypersensitivity during

Page 1 of 13
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Functional bowel disorders are commonly characterized
by altered motility of the gut as well as abdominal pain.
Patients with bowel disorders are known to have
decreased thresholds to pain for both visceral and somatic
stimuli [1]. Ritchie [2] reported that patients with irritable
bowel syndrome (IBS) experience colonic pain at lower
balloon distension pressures than controls following
colorectal distension (CRD). Pain associated with low
pressure balloon distension is thought to be indicative of
visceral hypersensitivity caused by abnormal motility pat-
terns in patients with bowel disorders. The molecular
mechanism that is responsible for visceral hypersensitivity
is still currently unknown; however, it is thought to be
caused by changes in neuronal excitability of visceral
afferents [1].

To date, it is thought that visceral hypersensitivity is medi-
ated by sensory nociceptors of the dorsal root ganglia
(DRG) that transmit signals from the gut to the lumbosac-
ral spinal cord. Studies have shown a change in expression
of SP and CGRP [3-6] as well as the activation of the
NMDA receptor in response to inflammation [7-12]
within DRG neurons as well as spinal neurons. However,
Zhou et al. [12] reported that the N1 and C1 cassettes of
the NMDA receptor NR1 subunit are up-regulated in the
colon following 2,4,6-Trinitrobenzenesulfonic acid
(TNBS)-induced inflammation. It was hypothesized that
this up-regulation is involved in both visceral and somatic
hypersensitivity [13,14]. In addition, enteric neurons
expressing the proteinase-activated receptor- 2 (PAR2)
receptor were found to be involved in visceral hypersensi-
tivity. Coelho et al. [15] demonstrated that by administer-
ing the PAR2 specific agonist trypsin hyperalgesia
occurred in response to CRD in rats. Furthermore, Cenac

et al [16] reported that activation of the PAR2 receptor by
proteases generated hypersensitivity symptoms in IBS

Recently we demonstrated that a unique set of enteric
neurons, colospinal afferent neurons (CANs), co-localize
with several known nociceptive peptides and receptors
[17]. The aim of the current study was to determine if
CANs contribute to visceral hypersensitivity following
TNBS-induced inflammation. To date, studies have
shown that the transient receptor potential vanilloid
receptor 1 (TRPV1) has a functional role in visceral hyper-
sensitivity [18,19]. In our previous study we demon-
strated that the TRPV1 receptor was not expressed in
CAN[17]. In the present study we used resiniferatoxin
(RTX) to lesion TRPV1 spinal primary afferent innervation
to the colon to determine if the lesion could suppress
hypersensitivity to CRD.

CANs co-localize with neuron specific sodium channel
We previously reported that CANs are putative sensory
neurons within the enteric nervous system. It was hypoth-
esized that CANs are involved in nociception as demon-
strated by immunohistochemical characterization [17]. In
order to determine whether CANs are putative nociceptive
neurons, we performed immunohistochemistry (n = 5)
using an antibody raised against Na,1.9. Co-localization
demonstrated that 86% of CANs express the sodium
channel Na,1.9 (Fig 1). In conjunction with our previous
study [17], the data suggests that CANs are putative noci-
ceptive neurons.

Figure I
CANs express TTX-resistant sodium channel NaI,.9. Photomicrographs of Dil labelled neurons in the submucosal
plexus of colon showing co-localization with known nociceptive sodium channel Na, 1.9. Scale bars = 20 im.

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Molecular Pain 2009, 5:54

RTX treatment
Our data show that CANs are sensory neurons that may be
involved in nociception. Therefore, we wanted to deter-
mine if CANs contribute to visceral hypersensitivity. As
previously shown [17], CANs do not express TRPV1;
therefore, we used an intrathecal and colonic injection of
resiniferatoxin (RTX) to lesion TRPV1 expressing spinal
primary afferent innervation to the colon. Immunohisto-
chemical analysis revealed that all animals that received
RTX (n = 50) had minimal to no TRPV1 receptors present
in the colon (Fig 2a) or in DRG (L6-S1) neurons (Fig 2b).
Immunohistochemistry analysis confirmed that RTX (n =
5) did not disrupt the peptide and receptor profile of
CANs as compared to controls (n = 5) [17] (Fig 2c). Fur-
thermore, the thoracolumbar (T13-L1) region of spinal
cord has been implicated in visceral hypersensitivity [20-
23]. Immunohistochemical analysis revealed that all ani-
mals that received RTX had minimal to no TRPV1 recep-
tors present in the thoracic DRGs (T11-13) as compared
to controls (Fig 2d).

RTX decreases DRG afferent innervation of the colon
To verify the amount of remaining colonic innervation
from DRG neurons following RTX, we injected Dil into
the distal colon of both control (n = 5) and RTX animals
(n = 5) (Fig 3a). Cell counts determined that 87 + 12 total
DRG neurons per lumbosacral DRG innervate the colon
using this procedure. Following RTX treatment, we found
26 + 6 DRG neurons per lumbosacral DRG innervate the
colon. Thus, RTX treatment eliminates approximately
two-thirds of visceral afferent innervation to the colon (p
< 0.001) (Fig 3a). Since RTX treatment does not eliminate
all DRG innervation to the colon, we performed immuno-
histochemistry to verify RTX did lesion all TRPV1 express-
ing neurons. Cell counts verified that the remaining Dil
labelled DRG cells did not express TRPV1 (p < 0.0001)
(fig 3b). We also determined that the remaining Dil
labeled DRG neurons did not express the PAR2 receptor
(p < 0.0001) (fig 3c). This finding is in agreement with
data from Amadesi et al. [24] which demonstrated that
the PAR2 receptor co-localized with TRPV1 receptors in
DRG neurons. Further analysis revealed that the neuroki-
nin-1 (NK1) receptor was still present on the remaining
Dil neurons (fig 3d).

All rats that received TNBS enemas (n = 30) showed char-
acteristics of inflammation: infiltration of neutrophils, an
increase in the number of mast cells and ulceration of the
mucosa 14 days post TNBS inflammation. Control ani-
mals showed a limited amount of neutrophil infiltration
and no ulcerations of the mucosa. (Fig 4a).

Behavior testing
We measured both visceral and peripheral mechanical
hypersensitivity at 14 days post-TNBS inflammation. We
observed a decrease in balloon pressure threshold for
both non-RTX (n = 10; p < 0.001) and RTX TNBS animals
(n = 10; p < 0.001) (Fig 4b). However, for peripheral
mechanical threshold of the hindpaw we observed a
decrease in threshold for non-RTX TNBS animals only (n
= 10; p < 0.05), as compared to controls (n = 10; p > 0.05)
(Fig 4b).

TNBS-induced inflammation
As previously reported, CANs co-localize with the NR1
and NR2D subunits of the NMDA receptor as well as with
the PAR2 receptor [17]. However, in control animals a
minimal number of CANs co-localize with the N1 and C1
splice variants of the NR1 subunit of the NMDA receptor
(Fig 5a, b). Fourteen days post-TNBS inflammation, both
the N1 and C1 splice variants were up-regulated, a 50%
(10% 1.145 control; 70% 3.3 TNBS) and 51% (8% +
8.6 control; 59.8% + 1.7 TNBS) increase in co-localization
with CANs respectively (Fig 5). This is in agreement with
another study that found an up-regulation of the NR1
splice variants in the colon following TNBS inflammation
[12]. Furthermore, the co-localization of Dil with the
PAR2 receptor appeared to decrease by approximately
50% following inflammation (P < 0.0003) (Fig 6a, b). The
antibody raised against the PAR2 receptor recognizes the
N-terminal end of the receptor or the un-activated form. A
commercial antibody for the C-terminal end of the PAR2
receptor in rat is currently unavailable. We therefore used
RT-PCR to detect PAR2 receptor mRNA, and found that
the PAR2 receptor is not down-regulated following TNBS
induced inflammation (Fig 6c, d). Therefore, the decrease
in co-localization in CANs suggests the receptors were
activated by proteases released from mast cells following
inflammation of the colon. Immunohistochemical analy-
sis determined, 14 days post-inflammation, co-localiza-
tion of Dil labelled cells with SP decreased approximately
60% as compared to controls (P < 0.0008) (Fig 6e). The
data suggests that following inflammation the PAR2
receptor expressed on CANs is activated thus causing the
release of SP.

CANs are putative nociceptors
The present results suggest that CANs are putative nocice-
ptive neurons that may contribute to visceral hypersensi-
tivity. We demonstrated that CANs co-localize with
Na,1.9, a sodium channel that is present on nociceptive
DRG neurons as well as enteric neurons [25,26]. Although
the presence of Na,1.9 expressed on neurons does not
determine if the neuron is a nociceptor, the presence of
Na,1.9 is highly correlated with neurons that are found to
be nociceptive [27]. Treatment with RTX resulted in the

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Molecular Pain 2009, 5:54

a a'


b b'







Figure 2
Expression of the TRPV I receptor is eliminated following treatment with RTX. Photomicrographs of Dil and
TRPV I expression in control(a') and RTX(a") lesioned rats in submucosal plexus of the colon. RTX results in a decrease in the
expression of TRPVI labeling in the colon. Photomicrographs that show a decrease in both Dil and TRPVI expression in lum-
bosacral DRG neurons(b") as compared to controls(b'). (c) Immunohistochemical characterization of CANs following RTX
lesioning showed no change in peptide and receptor expression as compared to controls. RTX results in a decrease in the
expression of TRPVI labeling in thoracolumbar DRG neurons (e). Scale bars = 20 |pm(a) and 50 |pm(b, e).

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Molecular Pain 2009, 5:54







U.PV1/-ontrol I VITI


"L 75-
c2oC 501
2 25-




-a. 50-

S 25-



2 75.
"-a 50'
- 25-


U.1onrl NK/T

Figure 3
Colonic innervation from lumbosacral DRG neurons is reduced following RTX treatment. (a)Application of the
retrograde tracer Dil into the colon wall revealed a 70% decrease in the number of DRG neurons that innervate colon as com-
pared to controls (control 87 12;RTX 26 6;n = 5, p < 0.001). Dil neurons no longer co-localized with the TRPV I recep-
tor(b) (p < 0.000 I) or the PAR2 receptor(c) (p < 0.000 I) following RTX lesioning. However, there was no change in the
expression of the NKI receptor (d). Scale bars = 50 inm.

lesioning of TRPV1-expressing DRG neurons of the lum-
bosacral and thoracolumbar regions of the spinal cord.
RTX also eliminated 70% of the lumbosacral DRG inner-
vation to the colon. However, RTX treatment did not elim-
inate the visceral hypersensitivity response in TNBS
treated animals. Although not definitive, the data suggests
that CANs may contribute to visceral hypersensitivity. The
visceral hypersensitivity observed seems to be associated

with changes in both the NMDA and PAR2 receptors. We
determined that the N1 and C1 cassettes of the NR1 sub-
unit of the NMDA receptor are up-regulated in CANs 14
days following TNBS-induced inflammation. Also, TNBS
inflammation causes the activation of the PAR2 receptors
and this activation may cause the release of SP. Both these
receptors have been implicated in mediating the visceral
hypersensitivity response [27,28]. The present study dem-

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Molecular Pain 2009, 5:54






. fi M

Nociceptive Threshold m Control


o 20


30 *

Mechanical Threshold


Figure 4
TNBS induced visceral but not somatic hypersensitivity is still present following RTX treatment. The presence
of inflammation was seen in animals 14 days post-TNBS inflammation as shown by infiltration of neutrophils in the lamina pro-
pria, an increase in mast cells and mucosal ulceration (a). Fourteen days post-inflammation resulted in a decrease in balloon
threshold for both Non-RTX and RTX animals. However, a decrease in peripheral mechanical threshold was seen in non-RTX
animals but not RTX animals (b).

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Molecular Pain 2009, 5:54






3- 75-
= o0



n .s- I I


0- 75-

0 g 50-
w c 25-








Figure 5
NMDA receptor expression on CANs increases following TNBS-induced inflammation. The NMDA receptor NRI
cassettes NI (a) and Cl(c) are up-regulated in Dil labelled neurons 14 days post-inflammation. NI co-localization increased
50% (70% 3.3) as compared to controls (10% 1.145;p < 0.0001) (b). Where as Cl co-localization increased 51% (59.8%
1.7) as compared to controls (8% 8.6;p < 0.001) (d). Scale bars = 20 im.

onstrates that CANs are putative nociceptive neurons that
are involved in the plasticity of the colon following
inflammation. This and our previous study [17] further
suggest that sensory information can be transmitted from
the gut to the spinal cord by CANs.

Visceral hypersensitivity present following RTX treatment
Recently studies have suggested that the TRPV1 receptor is
involved in the visceral hypersensitivity response [18,19].
However, our current study demonstrates that elimina-
tion of the TRPV1 receptor expressing neurons does not
alter the visceral hypersensitivity response to colorectal
distension (CRD). It is important to note that our study
looks at visceral hypersensitivity 14 days following
inflammation. Some studies looking at visceral hypersen-

sitivity have only studied this effect using acute inflamma-
tory agents that do not last longer than 8 days [29-31].
Conversely, Miranda et al. [32] demonstrated that appli-
cation of a TRPV1 antagonist (JYL1421) 14 days following
TNBS inflammation resulted in an attenuation of the vis-
ceral motor response (VMR) as compared to the TNBS-
only group. However, the response was still present as
compared to control responses. Thus, the data suggest that
blocking the TRPV1 receptor does result in a decrease in
visceral hypersensitivity but does not eliminate the
response entirely. The authors hypothesized that TRPV1
may be involved in the initial inflammation response and
hypersensitivity but not the maintenance of the response.
Another study has implicated the TRPV1 receptor in
motility. De Schepper et al. [33] used In-Vivo techniques

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Molecular Pain 2009, 5:54









e 100-

8 75-
5 "
B 25
0- --


Control- Balloon TNBS + Balloon


Figure 6
Activation of the PAR2 receptor expressed on CANs following TNBS induced inflammation. Approximately 40%
of CANs expressing the PAR2 receptor are activated following inflammation as shown by an antibody that detects the N-ter-
minal end of the PAR2 receptor (a, b) (p < 0.0003). Typical RT-PCR products for PAR2 (598 bp) and GAPDH (720 bp) (c). RT-
PCR analysis revealed that mRNA expression of PAR2 does not change due to TNBS induced inflammation as compared to
controls (c, d). A 60% decrease in the co-localization of SP expressed on CANs was seen following inflammation as compared
to controls (p < 0.0008) (e). Scale bars = 20 rm.

in conjunction with capsazepine, a TRPV1 antagonist, to
demonstrate that the sensitization of the TRPV1 receptor
inhibits colitis-induced abnormal motility patterns.
Therefore, it is plausible that the TRPV1 receptor is
involved in an extrinsic reflex pathway modulating motil-
ity patterns during a state of inflammation. It has also
been shown that this abnormal motility reflex was elimi-
nated following ligation of the pelvic nerve [34]. In addi-
tion, Jones, III et al. [19] demonstrated that the TRPV1
receptor may be involved in non-inflammatory visceral
hypersensitivity using a TRPV1 knock-out model. The
authors demonstrated that TRPV1 -/- mice receiving
zymosan and CRD did not show an increase in the VMR
response; however, matched controls did have an increase
in the VMR response. In this model zymosan did not
induce inflammation, as shown by an MPO assay, which
is in contrast with studies done in rats showing that

zymosan causes inflammation [29]. Thus, our data in con-
junction with the studies mentioned above suggest that
the TRPV1 receptor may be involved in the mechanical
component of the colonic inflammatory response but
may not be the major component to maintaining visceral

Furthermore, it is important to note that the present study
only looks at the involvement of the lumbosacral region
of the spinal cord and its role in visceral hypersensitivity.
Other studies have implicated the thoracolumbar region
of the spinal cord in the mediation of the visceral hyper-
sensitivity response [20-23,23]. Traub et al. [20] demon-
strated that both lumbosacral and thoracolumbar regions
of the spinal cord were activated during CRD as shown by
cFos and clun labeling. However, the sacral region of the
spinal cord had a higher cFos and cJun response to CRD

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GAPDH 720b]
PAR2 600bl

GAPDH 720t
PAR2 600t

Molecular Pain 2009, 5:54

(80 mmHg) compared to the thoracic region. A later study
indicated that there is a differential involvement of the
two regions of the spinal cord following CRD [23]. To
date, studies have not shown what receptors are involved
in thoracolumbar mediation of visceral hypersensitivity
and the exact role of this region in visceral hypersensitivity
has yet to be determined. In the current study, we have
shown that RTX lesions TRPV1 expressing DRG neurons
in the thoracolumbar region in addition to the lumbosac-
ral region following an intrathecal injection at the L6/S1
region of spinal cord and an intracolonic injection. There-
fore, if the thoracolumbar DRG neurons are involved in
visceral hypersensitivity, the present study demonstrates
that the TRPV1 expressing neurons are not necessary to
mediate visceral hypersensitivity.

Up regulation of NMDA receptors
The NMDA receptor expressed on DRG neurons has com-
monly been thought to regulate visceral hypersensitivity.
In addition, the NMDA receptor is expressed on enteric
neurons of the colon in both rats and humans [35,36]. It
was found that the NR2 subunits of the NMDA receptor,
NR2B and NR2D, are present on colonic neurons [37].
Furthermore, Zhou et al. [12] showed the NR1 cassettes,
N1 and C1, were up-regulated in a sub-set of myenteric
neurons in the colon following TNBS-induced inflamma-
tion. In the present study we found similar results; how-
ever, we found that both the N1 and C1 cassettes were up-
regulated in submucosal CANs. Studies have shown that
when the N1 insert is present in NR1 the current ampli-
tude is increased. This effect was shown to be potentiated
by Zn2+ and spermine [38,39]. Furthermore, Zheng et al
[39] hypothesized that the increase in agonist-evoked cur-
rents may be a result of a conformational change induced
by the expression of N1. This data suggests that an
increase in NMDA receptor activity due to the increased
expression of the N1 cassette on CANs may be contribut-
ing to the visceral hypersensitivity response observed fol-
lowing RTX and TNBS treatment. We also observed the
presence of the C1 cassette on CANs following inflamma-
tion. The C1 insert is known to have an endoplasmic retic-
ulum (ER) retention signal as well as four series for
phosphorlation. It is thought C1 is involved in the traf-
ficking of newly formed NMDA receptors [40]. Therefore,
the presence of both the N1 and C1 cassettes following
inflammation could increase the activity of NMDA recep-
tors on CANs causing neuroplasticity in the colon.

Activation of PAR2
Recently it was suggested that activation of the PAR2
receptor is involved in inflammation-induced visceral
hypersensitivity [16,41-43]. In the current study we dem-
onstrated that the N-terminal end of PAR2 receptors on
CANs was no longer present following TNBS-induced
inflammation. It is known that proteases released in the

colon during states of inflammation cleave the N-terminal
end of the PAR2 receptor [41,44]. Our antibody for PAR2
was raised against the amino acid sequence on the N-ter-
minal end of the PAR2 receptor just prior to the cleavage
site; thus, the decrease in co-localization translates to an
activation of the receptor. Previous studies looking at the
role of proteases in IBS patients demonstrated that the
activation of the PAR2 receptor can generate hypersensi-
tivity symptoms in IBS patients [16]. In this study, colonic
biopsies from IBS patients were found to have elevated
levels of the proteases trypsin and tryptase as compared to
non-IBS patients. A study using rats concluded that by
administering the PAR2 specific agonist trypsin, hypersen-
sitivity occurred in response to colorectal distention [15].
Hyun et al. [43] demonstrated that PAR2 -/- mice showed
a decrease in signs of inflammation following TNBS-
induced inflammation as shown by H&E staining, mye-
loperoxidase (MPO) activity, macroscopic damage score,
and histological analysis. Furthermore, inflammatory
mediators such as intracellular adhesion molecule-1
(ICAM-1) and vascular cell adhesion molecule (VCAM-1)
were decreased and cyclooxygenase-1 (COX-1) was
increased in PAR2-/- mice, suggesting that PAR2 receptors
play a pro-inflammatory role. Previous studies suggest
that activation of the PAR2 receptor causes the release of
SP [41,42] SP is known to bind NK1 receptors in the dor-
sal horn of the spinal cord suggesting this interaction aids
in mediating hypersensitivity[6]. Amadesi et al. [24] dem-
onstrated that DRG neurons expressing the PAR2 receptor
also express the TRPV1 receptor. The authors determined
that activation of the PAR2 receptor caused release of SP
following capsaicin injections. In the current study we
demonstrated that lesioning of DRG neurons expressing
TRPV1 receptors also eliminated expression of the PAR2
receptor in DRG neurons that innervate the colon. How-
ever, following inflammation, the remaining PAR2 recep-
tors in the colon were activated and the visceral
hypersensitivity response was present. Since we know that
activation of the PAR2 receptor causes the release of SP,
the data suggests that activation of PAR2 receptors on
CANs may mediate the visceral hypersensitivity response
via the release of SP acting on the remaining NK1 recep-
tors expressed on DRG neurons. Further studies are
needed to delineate the exact mechanism of this hyper-
sensitivity response and whether SP is involved.

In conclusion, the present study found that CANs are
putative nociceptors as determined by the expression of
Navl.9 as well as other peptides and receptors commonly
found on nociceptive neurons as shown previously [17].
We demonstrated that visceral hypersensitivity is still
present in inflamed animals when TRPV1 expressing DRG
neurons are eliminated. Following inflammation, CANs
express the activated form of both NMDA and PAR2

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Molecular Pain 2009, 5:54

receptors. The activation of these receptors may induce
neuroplasticity in the colon which, in turn, may contrib-
ute to the visceral hypersensitivity response.

Experiments were performed on male Sprague-Dawley
rats (n = 105) weighing 200-250 g. They were housed in
pairs with free access to food and water in the University
of Florida's animal care facility with a 12-h light/dark
cycle. These facilities are AAALAC accredited. All experi-
ments conformed to guidelines on the ethical use of ani-
mals as published by the International Association for the
Study of Pain. All procedures were reviewed and approved
by the University of Florida Institutional Animal Care and
Use Committee.

Resiniferatoxin (RTX) injections
Prior to injection, the animals were anesthetized with iso-
flurane (1-3% in 02). For intrathecal injections, an 18-
gauge needle was used to make a lumbar puncture
between vertebrae L1/L2. A small plastic tube (PE-10; Bec-
ton Dickinson, Sparks, MD) was inserted through the nee-
dle and the RTX solution (200 ng/25 Al in PBS) was
injected slowly over 2 minutes. Following intrathecal
injection, animals received an enema of RTX. A small plas-
tic tube (PE-160; Becton Dickinson, Sparks, MD) was
inserted 7 cm through the anus and the RTX solution (200
ng/500 il) was infused slowly over 5 minutes. Animals
were allowed to recover for two weeks prior to further pro-

2,4,6-trinitrobenzene sulfonic acid (TNBS) enema
Prior to the enema, the animals were anesthetized with
isoflurane (1-3% in 02). Animals received enemas of
either TNBS (20 mg) in 50% ethanol and saline (1 ml) or
saline alone (1 ml). A small plastic tube (PE-160; Becton
Dickinson, Sparks, MD) was inserted through the anus 7
cm and the solution was infused slowly over 5 minutes.

Laminectomy and Dil labeling
Prior to laminectomy, the animals were anesthetized with
isoflurane (1-3% in 02). The dorsal portion of the L1 and/
or L2 vertebrae was removed to expose the L6 and S1 seg-
ments of spinal cord. The dura covering the spinal cord
was removed and a 5 x 2 mm piece of gel foam (Henry
Schien, Melville, NY, USA) was placed onto the dorsal sur-
face of the spinal cord. The gel foam was soaked in 40 1il
of the retrograde tracer 1,1'-dioctadecyl-3,3,3',3'-tetrame-
thyl-indo-carbocyanine perchlorate (Dil) (Molecular
Probes, Eugene, OR, USA; 2.5 mg/ml in DMSO). The
wound was closed by suturing in layers. A triple antibiotic
was applied to the wound site and buprenorphine (0.3
mg/kg) was given i.m. twice a day for approximately 3
days after surgery.

Laparotomy and Dil labeling of colonic DRG neurons
Prior to laparotomy, the animals were anesthetized with
isoflurane (1-3% in 02). A small incision was made in the
abdomen to expose the colon. Ten Dil injections (2 Al per
injection; total 20 til) were made in the wall of the colon
using a Hamilton syringe, approximately 2 mm proximal
to the bladder moving orally. The wound was closed by
suturing in layers. A triple antibiotic was applied to the
wound site and buprenorphine (0.3 mg/kg) was given
i.m. twice a day for approximately 3 days after surgery.

Behavior testing
For all behavioral testing, the researcher was blinded to all
treatment groups.

Peripheral mechanical stimulation
Mechanical hypersensitivity was measured using an auto-
matic Von Frey device (dynamic plantar aesthesiometer
(Ugo Basile Biological Research Apparatus, Italy). Ani-
mals were placed within a plastic enclosure on a wire
mesh floor. A computer-driven filament was extended up
through the mesh floor and exerted an increasing amount
of pressure (0-50 g) onto the hind paw. Both hind paws
were tested. Mechanical pain threshold was determined
by the amount of force required for the animal to with-
draw its hind paw. The stimulus was repeated four times
with 5 minute intervals and the mean pressures at the
mechanical threshold were recorded for each rat.

Colonic distension
While anesthetized with isoflurane (1-3% 02) a balloon
(7 cm in length) attached to small plastic tubing (PE-160;
Becton Dickinson, Sparks, MD) was inserted through the
anus and secured in place by taping the tubing to the base
of the tail. Rats were placed in a plastic restraining device
in order to prevent the animals from removing the bal-
loon. The tubing was connected to a pressure transducer
(Harvard Apparatus, Holliston, Massachusetts) to per-
form colorectal distension (CRD). Following recovery
from anesthesia, the animals were allowed 10 minutes to
acclimatize before behavioral testing began. The rats then
received distension of the colon in 10 mmHg intervals (0-
80 mmHg) until the first contraction of the testicles, tail,
or abdominal musculature occurred. This is described as
the visceral pain threshold indicative of the first nocicep-
tive response. CRD was repeated four times with 5 minute
intervals and the mean pressures at the nociceptive thresh-
old were recorded for each rat.

Perfusion fixation
After a survival time of 10 days (for surgery animals) or
the end point of the experiment, animals were given a
lethal dose of pentobarbital i.p. and perfused through the
heart with cold 0.9% saline followed immediately with
cold 4% paraformaldehyde in phosphate buffered saline

Page 10 of 13
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Molecular Pain 2009, 5:54

(PBS). After fixation, the spinal cord, dorsal root ganglia
(DRG) (L6-S1 and T11-L1) and the colon of the animal
were removed. Tissue was then post-fixed in 4% parafor-
maldehyde in PBS for 24 hours at 40C and then stored in
30% sucrose at 40C for at least 24 hours.

Hematoxylin and eosin staining
Colonic tissue from all behavioral animals was processed
for histopathological evaluation. The tissue was processed
using standard techniques for hematoxylin and eosin
staining. The tissue was then evaluated for signs of colitis:
infiltration of neutrophils in the lamina propria, an
increase in mast cells and mucosal ulceration.

Colonic tissue was sectioned at 20 gm and DRG tissue was
sectioned at 10 gm on a cryostat, cut in 1:5 series sections,
and air-dried for 1 hour. All preparations were washed 3
times (10 minutes each) in PBS, placed in blocking buffer

containing 3% Normal Goat Serum (NGS) with PBS for 1
hour, and incubated in primary antibody in 3% NGS/
0.3% tween-20/PBS (table 1) for 24 hours at 4C. The sec-
tions were then washed 3 times in PBS (10 minutes each)
followed by a 1 hour incubation in secondary antibody
Alexa Fluro 488 (1:1000; Molecular Probes, Boston, MA)
in 3%NGS/0.3% tween-20/PBS (table 2). Tissue was then
washed 3 times (10 minutes each) and coversliped with
ProLong Antifade Kit mounting media (Molecular
Probes, Boston, MA) or Vectashield mounting media
(Vector Laboratories, Burlingame, CA). The sections were
visualized with filters for red and green excitation. Images
were photographed on a Leica DM LB2 Fluorescence
microscope (Leica, Wetzlar, Germany). Negative controls,
where the secondary was applied to the sections in the
absence of primary antibodies, were used to verify that
non-specific binding of secondary antibodies to tissue did
not occur. All images were processed using the Adobe
Photoshop program. For colonic cell counts, at least a

Table I: Primary antibodies used in immunohistochemistry of CANs.













Goat conjugated to IB4







Goat anti-Rabbit

Donkey anti-Mouse

Goat anti-chicken





BD Biosciences

BD Biosciences

Santa Cruz



Aves Lab, Inc.

Affinity Bioreagents

Courtesy of Dr. Michael ladarola





Courtesy of Dr. Michael ladarola

Courtesy of Dr. Michael ladarola




Page 11 of 13
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NF 200

Alexa Fluor 488

Alexa Fluor 488

Alexa Fluor 488

Molecular Pain 2009, 5:54

Table 2: RT-PCR primer sequences

Primer name Forward Sequence

Temperature (C)

Reverse Sequence

Temperature (C)

Product Length (bp)




total of 100 cells per immunolabel were counted for anal-
ysis. For DRG cell counts, all Dil labelled cells were
counted per DRG. Cell counts were done using the ImageJ
program (NIH, Bethesda, MD) where Dil positive cells (in
red) were counted and marked; double labelled cells were
determined by superimposing the marked image with the
label being investigated (in green).

Animals were euthanized with CO2 and the colon was
quickly removed. Total RNA was isolated from colonic tis-
sues using RNeasy Mini Kit from Qiagen (Valencia, CA,
USA). Target transcripts were amplified with PCR primers
from GenoMechanix (Gainesville, FL, USA) listed in Table
2. RT-PCR reactions were carried out using Access RT-PCR
System from Promega (Madison, WI, USA) with the fol-
lowing cycle conditions: Initial denaturation at 95 C for
2 minutes; denaturing at 950C for 30 seconds; annealing
at 55 C for 1 minute and extension at 72 C for 2 minutes.
A total of 32 cycles was used, followed by a final extension
step of 720 C for 5 minutes. PCR products were separated
on 1.2% agarose gel with lx TBE buffer, viewed with
ethidium bromide and analyzed with Bio-Rad Gel Doc
EQ Gel Documentation System, Bio-Rad Laboratories
(Hercules, CA, USA).

Statistical analysis
All statistics were run using Prism GraphPad version 6. For
analysis of co-localization an unpaired t-test was used. For
analysis of behavioral testing a two-way analysis of vari-
ance (ANOVA) followed by Bonferroni posttest was used
for each time point. A one-way ANOVA was used to ana-
lyze RT-PCR data. All values are expressed as means +
SEM. Data sets were considered significant for P < 0.05.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
SKS participated in the design of the study, carried out all
of the experiments and analyses and helped write the
manuscript. RMC guided the design of the study and
helped with writing the manuscript. All authors read and
approved the final manuscript.


The authors thank Dr. Michael ladarola, from the National Institute of Den-
tal and Craniofacial Research (NIDCR) in Bethesda, MD for supplying the
NRI splice variants specific antibodies. We would like to thank Dr. John
Neubert from the Department of Orthodontics at the University of Florida
for supplying the resiniferatoxin. The work summarized here was sup-
ported by the National Institutes of Health NS045614

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