Identification of the visceral pain pathway activated by noxious colorectal distension in mice.

Abstract

In patients with irritable bowel syndrome, visceral pain is evoked more readily following distension of the colorectum. However, the identity of extrinsic afferent nerve pathway that detects and transmits visceral pain from the colorectum to the spinal cord is unclear. In this study, we identified which extrinsic nerve pathway(s) underlies nociception from the colorectum to the spinal cord of rodents. Electromyogram recordings were made from the transverse oblique abdominal muscles in anesthetized wild type (C57BL/6) mice and acute noxious intraluminal distension stimuli (100-120 mmHg) were applied to the terminal 15 mm of colorectum to activate visceromotor responses (VMRs). Lesioning the lumbar colonic nerves in vivo had no detectable effect on the VMRs evoked by colorectal distension. Also, lesions applied to the right or left hypogastric nerves failed to reduce VMRs. However, lesions applied to both left and right branches of the rectal nerves abolished VMRs, regardless of whether the lumbar colonic or hypogastric nerves were severed. Electrical stimulation applied to either the lumbar colonic or hypogastric nerves in vivo, failed to elicit a VMR. In contrast, electrical stimulation (2-5 Hz, 0.4 ms, 60 V) applied to the rectum reliably elicited VMRs, which were abolished by selective lesioning of the rectal nerves. DiI retrograde labeling from the colorectum (injection sites 9-15 mm from the anus, measured in unstretched preparations) labeled sensory neurons primarily in dorsal root ganglia (DRG) of the lumbosacral region of the spinal cord (L6-S1). In contrast, injection of DiI into the mid to proximal colon (injection sites 30-75 mm from the anus, measured in unstretched preparations) labeled sensory neurons in DRG primarily of the lower thoracic level (T6-L2) of the spinal cord. The visceral pain pathway activated by acute noxious distension of the terminal 15 mm of mouse colorectum is transmitted predominantly, if not solely, through rectal/pelvic afferent nerve fibers to the spinal cord. The sensory neurons of this spinal afferent pathway lie primarily in the lumbosacral region of the spinal cord, between L6 and S1.

Full text

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www.frontiersin.org February 2011 | Volume 5 | Article 16 | 1
Original research article
published: 22 February 2011
doi: 10.3389/fnins.2011.00016
Identification of the visceral pain pathway activated by
noxious colorectal distension in mice
Melinda Kyloh, Sarah Nicholas, Vladimir P . Zagorodnyuk, Simon J. Brookes and Nick J. Spencer*
Department of Human Physiology, Flinders Medical Science and Technology Cluster, Flinders University, Adelaide, SA, Australia
In patients with irritable bowel syndrome, visceral pain is evoked more readily following distension
of the colorectum. However, the identity of extrinsic afferent nerve pathway that detects and
transmits visceral pain from the colorectum to the spinal cord is unclear. In this study, we
identified which extrinsic nerve pathway(s) underlies nociception from the colorectum to the
spinal cord of rodents. Electromyogram recordings were made from the transverse oblique
abdominal muscles in anesthetized wild type (C57BL/6) mice and acute noxious intraluminal
distension stimuli (100–120 mmHg) were applied to the terminal 15 mm of colorectum to
activate visceromotor responses (VMRs). Lesioning the lumbar colonic nerves in vivo had no
detectable effect on the VMRs evoked by colorectal distension. Also, lesions applied to the right
or left hypogastric nerves failed to reduce VMRs. However, lesions applied to both left and right
branches of the rectal nerves abolished VMRs, regardless of whether the lumbar colonic or
hypogastric nerves were severed. Electrical stimulation applied to either the lumbar colonic or
hypogastric nerves in vivo, failed to elicit a VMR. In contrast, electrical stimulation (2–5 Hz, 0.4 ms,
60 V) applied to the rectum reliably elicited VMRs, which were abolished by selective lesioning
of the rectal nerves. DiI retrograde labeling from the colorectum (injection sites 9–15 mm from
the anus, measured in unstretched preparations) labeled sensory neurons primarily in dorsal
root ganglia (DRG) of the lumbosacral region of the spinal cord (L6-S1). In contrast, injection
of DiI into the mid to proximal colon (injection sites 30–75 mm from the anus, measured in
unstretched preparations) labeled sensory neurons in DRG primarily of the lower thoracic level
(T6-L2) of the spinal cord. The visceral pain pathway activated by acute noxious distension of
the terminal 15 mm of mouse colorectum is transmitted predominantly, if not solely, through
rectal/pelvic afferent nerve fibers to the spinal cord. The sensory neurons of this spinal afferent
pathway lie primarily in the lumbosacral region of the spinal cord, between L6 and S1.
Keywords: sensory neuron, pain, afferent, nociceptor
signals from the rectum to the spinal cord (Grundy, 2002, 2004;
Brierley et al., 2004; Furness, 2006; Spencer et al., 2008a; Song et
al., 2009). However, due to difficulties in identifying extrinsic nerve
tracks in small laboratory mammals such as mice, there have been
no lesion studies to identify unequivocally which extrinsic afferent
pathway(s) detect and transmit painful stimuli arising from the GI
tract to the spinal cord.
In mammals, two distinct spinal afferent nerve pathways
exist that can potentially transmit sensory information from the
rectum and distal colon to the spinal cord. These are known as
the lumbar colonic nerves (LCN)/lumbar splanchnic and sacral
colorectal/pelvic nerves (Brierley et al., 2004). However, which of
these pathways is important in the detection and transmission of
visceral pain from the colorectum is unclear. In mouse, these two
distinct sensory nerve pathways have been recently shown to pos-
sess at least five different classes of afferent fibers, each of which
responds selectively to different stimuli (Brierley et al., 2004). In
general, it is now known that the colorectal/pelvic pathway is a
predominantly low threshold sensory pathway (Brierley et al.,
2004; Spencer et al., 2008b; Feng et al., 2010), responding to low
intensity mechanical stimulation, whereas the lumbar splanchnic
IntroductIon
Patients with irritable bowel syndrome (IBS) have reduced thresh-
old of pain and discomfort in the colon and rectum (Mertz et al.,
1995). There is now abundant evidence that extrinsic spinal afferent
nerves must transmit these nociceptive signals arising from the
gastrointestinal (GI) tract to the spinal cord. However, there are a
number of extrinsic nerve pathways that could potentially transmit
nociceptive signals from the colorectum to the spinal cord and
the contribution of these pathway(s) to transmission of nocicep-
tive stimuli is currently unknown. If we wish to make progress in
developing therapies that target colorectal pain, it is essential that
we identify which sensory nerves detect noxious stimuli arising
from the colorectum and the precise pathway that these afferents
take in reaching the spinal cord.
Rodents are the most commonly used models to study mecha-
nisms underlying visceral pain in the GI tract, because they reliably
generate a stereotypical pain reflex in response to acute noxious
colorectal distension, known as the visceromotor responses (VMRs;
Larsson et al., 2003; Arvidsson et al., 2006). It is now clear that
spinal afferents, whose cell bodies lie in the dorsal root ganglia
(DRG) underlie the sensory pathway that conducts visceral pain
Edited by:
Paul P . Bertrand, University of
New South Wales, Australia
Reviewed by:
Christopher Keating, University of
Sheffield, UK
Bin Feng, University of Pittsburgh, USA
*Correspondence:
Nick J. Spencer, Department of Human
Physiology, Flinders University, GPO
Box 2100, Adelaide, SA 5001, Australia.
e-mail: nicholas.spencer@flinders.
edu.au

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Kyloh et al. Pain pathway activated by colorectal distension
(IMA) and colon. This thread was then raised vertically, such
that the distal colon was separated from the abdominal aorta by
a space of approximately 3–4 mm. This allowed sufficient space
to place fine transmural stimulating wires across the IMA and all
the LCN. It also allowed sufficient space to allow the LCN to be
severed with ultra fine surgical dissecting scissors. To access the
hypogastric nerves, the junction between the abdominal aorta
and left and right iliac arteries was identified (see Figure 1; Miller
and Szurszewski, 1994). The hypogastric nerves lie directly above
(dorsal) to the left and right iliac arteries. This allowed clear vis-
ualization of the hypogastric nerves to facilitate lesions of the
nerves, or allow direct access to stimulation by fine transmural
electrical stimulation. To directly stimulate the hypogastric nerves
using electrical stimulation, a fine 8/0 suture thread was looped
underneath each branch of the hypogastric nerves so that the
nerve trunk was geographically separated from the iliac artery.
This allowed transmural electrical stimulating wires to be placed
around the nerve and not apply electrical stimuli to the iliac arter-
ies themselves. To lesion the rectal nerves, a small incision was
made through the skin caudal to the pelvic bone. The rectum was
exposed and fine rectal nerves visualized. Once visualized, fine
surgical scissors were used to lesion all rectal nerves between the
anus and IMA.
dil retrograde tracIng
Mice of either sex were anesthetized with pentobarbital sodium
(200–300 μL of 6 mg/mL) and the depth of anesthesia was moni-
tored by lack of response to a hind limb or tail pinch. Upon
failure to elicit a tail or hind limb pinch, a midline laparotomy
was made to expose the abdominal cavity. The ileum and cecum
was gently reflected to one side of the abdominal cavity to expose
the colon. The retrograde neuronal tracer, 1,1′-didodecyl-3,3,3,3′-
tetramethylindocarbocyanine perchlorate (DiI C12 version), was
injected into two sites along the colon: one site was a distance
of 9–15 mm from the anus (when measured in unstretched
preparations). This equated to a distance of 19–30 mm from
the anus when the same segments of colorectum were removed
from mice, stretched, and pinned taught in a petri dish. The
second DiI injection site was made into the mid–proximal colon,
a distance of 30–75 mm from the anus (measured in unstretched
preparations), which equated to a distance of 60–100 mm from
the anus when the same preparations were stretched and pinned
taught. For DiI injections, a sterile 25 gauge needle connected to
a Hamilton syringe (5 μL maximum volume; Hamilton Company,
Reno, NV, USA) was used to penetrate through the serosal sur-
face. A series of two to four injection sites were made into the
colonic musculature, and a total DiI injection volume of 2–5 μL
was made. A fine layer of gauze was used to wrap underneath
the colon to prevent any leakage of DiI. The intestine and cecum
was carefully repositioned back into the abdominal cavity and
the abdominal muscles sutured closed. Mice were allowed to
recover for a period of 7 days following dye injections, at which
point mice were sacrificed. Following euthanasia, the colon and
abdominal viscera were inspected for leakage of DiI. Any prepara-
tions showing potential spread or leakage of DiI were discarded
from analysis. DRGs from bilateral spinal levels (T1-S3, inclusive)
were then removed. DRGs were fixed in paraformaldehyde (4%)
pathway is predominantly a high threshold pathway, carrying
only a small proportion of stretch sensitive (10%) afferent fibers
(Brierley et al., 2004).
In this study, we developed a novel in vivo preparation in which
it was possible, for the first time, to identify and lesion (or stimu-
late) the major extrinsic nerve pathways between the colorectum
and spinal cord, whilst eliciting VMRs to noxious colorectal dis-
tension. This allowed us to identify which extrinsic afferent nerve
pathway(s) are required for the generation of VMRs elicited fol-
lowing noxious colorectal distension. Secondly, we have injected
the retrograde neuronal tracer, DiI into a similar region of color-
ectum as distension stimuli were applied, to identify the location
of the sensory neurons that underlie the VMRs from this region.
We show that rectal/pelvic afferents are responsible for transmis-
sion of nociceptive information to the spinal cord following acute
noxious distension of the colorectum. The cell bodies of the DRG
sensory neurons in this pathway lie primarily in the lumbosacral
region of the spinal cord.
MaterIals and Methods
Protocol for actIvatIng vIsceroMotor resPonses
C57BL/6 wild type mice 3–6 months of age were anesthetized by
injection of pentobarbital sodium s.c. (200–300 μL of 6 mg/mL).
The depth of anesthesia was assessed by lack of response to hind
limb or tail pinch. A collapsible balloon was inserted 4–7 mm from
the anus and pressure was continuously monitored by a Gould pres-
sure transducer, connected to a Powerlab recording system using
Chart software (version 5.3, AD instruments, Sydney, Australia).
When fully inflated, the balloon measured 8 mm in length and
7 mm in width. Thus, the maximum length of bowel distended by
the balloon could be the terminal 15 mm of unstretched colorec-
tum. Electromyographic (EMG) electrodes were placed into the
left external oblique muscle and a reference electrode was placed
in the quadriceps muscle of the opposing leg. EMG recordings
were acquired at 20 kHz and recorded on a PC running LabChart
6 Pro software, high pass filtered (100 Hz), and analyzed using
Spike Histogram software (AD Instruments, Sydney, Australia).
Intraluminal pressure was increased by raising a column of water
from 0 to 100–120 mmHg and maintained for ∼15–20 s. In recent
studies (Zagorodnyuk et al., 2011), we have found VMRs can be
evoked in a near-linear fashion with increasing distension stimuli
up to 120 mmHg. Therefore, to be sure nerve lesions had prevented
all sensory signaling (both low and high threshold afferents) from
different nerve pathways, we applied stimuli up to 100–120 mmHg.
We were unable to detect any anatomical damage to the colorectum
following stimulation up to 120 mmHg. Stimuli above 200 mmHg
were required to induce damage.
technIque to lesIon extrInsIc sPInal nerve Pathways
For experiments in which lesions were made to each of the major
spinal nerve pathways, it was important to be able to visualize
these nerves in vivo. Once mice had been fully anesthetized, a
midline laparotomy was made to expose the internal organs.
The small intestine and cecum were gently removed from the
abdominal cavity, whilst retaining continuity with the vascular
and nervous supplies. A fine cotton thread was looped around
the distal colon at the junction of the inferior mesenteric artery

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Kyloh et al. Pain pathway activated by colorectal distension
results
IdentIfIcatIon of the PaIn Pathway underlyIng the vMr to
colorectal dIstensIon
In anesthetized mice, the abdominal cavity was exposed via mid-
line laparotomy to allow access to the lumbar colonic, hypogas-
tric nerves, and rectal nerves (Figure 1A). Nerve lesions were
then applied systematically to each of the major spinal affer-
ent pathways lying between the colorectum and spinal cord,
whilst applying acute noxious colorectal distension stimuli
(100–120 mmHg) to assess their contribution to the transmis-
sion of visceral pain arising from the colorectum. Once repro-
ducible VMRs were elicited (Figure 1B), a lesion was applied
through the LCN. Lesions of the LCN had no detectable effect
on the VMR to colorectal distension (Figures 1C,F, N = 9). In
these same animals in which the LCN had been severed, further
sectioning of the left and right hypogastric nerves also had no
effect on the VMRs to the same intensity stimulation (N = 5;
Figures 1D,F). However, further lesions applied to both left and
right branches of the rectal nerves always abolished VMRs, even
with maximal intensity distension (120 mmHg; Figures 1E,F,
N = 9). If lesions were applied to he rectal nerves, whilst pre-
serving the LCN and hypogastric nerves, no VMRs could be
elicited to colorectal distension (N = 3). This indicates that the
major sensory pathway activated by noxious distension of this
particular region of colorectum runs via the rectal and pelvic
nerves to the spinal cord.
for 3 days before cryoprotection in sucrose for 1 day. DRG were
cryostat-sectioned at −19°C and 12 μm sections made through-
out all ganglia. Two sections (sections 5 and 9) from each DRG
were retained and cover slip mounted. Fluorescent images of all
DRG sections were photographed within 2 days to avoid spread
and fading of DiI from filled nerve cell bodies. Because DRGs
are autofluorescent, DiI-labeled cell bodies were discriminated
by having fluorescence intensities three times above the back-
ground, which was defined by the mean fluorescence of neu-
ronal cell bodies obtained from similar age mice that had not
been injected with DiI. The mean number of labeled neurons
was counted from the two sections obtained from each of the
two DRGs obtained at each spinal segment (i.e., left and right
DRG). All experimentation described above was approved by
the Animal Welfare Committee at Flinders University of South
Australia (approval no. 640/07).
analysIs and statIstIcs
Data analysis
Electromyogram recordings of the mean firing rate of action poten-
tials was calculated during 10-s colorectal distensions. Results are
expressed as means ± SEM, with N referring to the number of
animals on which observations were made. Statistical analysis
was performed by ANOVA (one-way) using Prism v.5 software
(GraphPad Software, Inc., San Diego, CA, USA). Differences were
considered significant if P < 0.05.
Figure 1 | identification of the pain pathway underlying activation of
the visceromotor responses to colorectal distension. (A) Diagrammatic
representation of the major extrinsic nerve trunks that lie between the
rectum and spinal cord. (B) Control visceromotor responses elicited by acute
noxious colorectal distension. (C) Cutting only the lumbar colonic nerves did
not reduced the VMRs to acute noxious colorectal distension. (D) Similar
lesions applied to the hypogastric nerves also did not reduce the VMRs. (e)
However, cutting of the rectal nerves abolished the visceromotor responses.
(F) Shows the mean grouped data of VMRs evoked by acute noxious
colorectal distension after lesions applied to the lumbar colonic, hypogastric,
and rectal nerve trunks from N = 5 animals. * Refers to significant
difference (P < 0.05).

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Kyloh et al. Pain pathway activated by colorectal distension
from the anus (measured in unstretched preparations). These
same injection sites measured a distance of 19–30 mm in
stretched preparations. In control mice, it was found that the
greatest number of DRG neurons were located in the sacral
spinal cord at the level of L6 and S1, with a small proportion of
neurons labeled in T13-L1 (see Figure 3). On average, a mean
number of 17 ± 6 DRG neurons were labeled in L6 per section
(Figure 3, N = 5).
IdentIfIcatIon of the extrInsIc sensory InnervatIon of the MId
to ProxIMal colon
We also injected DiI further from the anus, in this case in the
region of the mid to proximal colon in a separate cohort. Injection
sites were 30–75 mm from the anus in unstretched preparations
(corresponding to a distance 60–100 mm in these same prepa-
rations stretched in vitro). The most striking difference was the
relatively low number of retrogradely labeled neurons from the
mid to proximal colon, despite the same volume of DiI injections
and similar number of injection sites into the muscularis externa.
The peak labeling of DRG was found to lie between T12 and L1
(N = 5), where the mean number of positively labeled neurons was
1.8 ± 0.7 at T13 (N = 5) and 0.8 ± 0.3 neurons positively labeled
at T9 (Figure 4, N = 5).
dIscussIon
It is well known that the colorectum of mammals receives a complex
extrinsic sensory innervation, via the lumbar splanchnic/lumbar
colonic and rectal/pelvic afferent pathways. Even within each of
these pathways, there are at least four or five different classes of
afferent fiber (Brierley et al., 2004), that respond to different types
of stimuli and different intensities of stimulation. Understanding
which nerve pathway(s) transmit visceral pain from the colorec-
tum to the spinal cord is of supreme importance, if we wish to
electrIcal stIMulatIon of the extrInsIc nerves InnervatIng
the colorectuM
We investigated whether transmural electrical stimulation applied
to the extrinsic nerves between the colorectum and spinal cord
would be able to elicit VMRs from each of the major nerve path-
ways. When fine transmural electrical stimulating wires were
placed around the lumbar colonic or hypogastric nerves, no VMRs
were evoked (5 Hz, 0.4 ms, 60 V, 5 s, N = 6). However, when the
same stimulating electrodes were positioned around the exposed
colorectum (5 mm from the anus), VMRs were reliably elicited
(Figure 2A). Lesions applied to the rectal nerves abolished VMRs
evoked by nerve stimulation (N = 5; Figure 2B). To confirm in these
preparations that rectal nerve lesions were specific for the rectal
nerves only, and had not prevented transmission along the spinal
cord, we applied a tail pinch and hind limb, after the rectal nerves
had been severed. In these cases, it was always possible to reliably
evoke VMRs following tail pinch or hind limb pinch, when the
rectal nerves had been severed and responses to colorectal stimula-
tion abolished (N = 4; Figure 2).
IdentIfIcatIon of the extrInsIc sensory InnervatIon of the
dIstal colon
We paid particular attention to the site at which DiI was injected
into the GI tract, measured as the distance from the anus in
both unstretched and stretched segments of isolated colorec-
tum. It was obvious that the distance at which DiI was injected
from the anus changed considerably depending on whether the
preparation was unstretched, or stretched and pinned taught in
a dissecting dish.
We injected the retrograde neuronal tracer, DiI, into a similar
region of colorectum that was distended by the intraluminal
balloon. Following laparotomy, DiI was injected through the
serosa and into the muscularis externa a distance of 9–15 mm
Figure 2 | Lesions to the rectal nerves selectively ablated responses to
colorectal distension and electrical stimulation of the colorectum. (A) In an
anesthetized mouse, a single hind limb pinch, tail pinch, electrical stimulation
(5 Hz, 0.4 ms, 60 V, 5 s) of the colorectum or acute noxious colorectal distension
all elicited VMRs. (B) Lesions applied only to the left and right branches to the
rectal nerves abolished VMRs to acute noxious colorectal distension and
electrical stimulation of the rectum, but not those evoked by hind limb pinch or
tail pinch.

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Kyloh et al. Pain pathway activated by colorectal distension
spinal cord, with only a small proportion from the thoracic spinal
cord. This observation is highly consistent with the results from
our lesion experiments which showed that lesions to the lumbar
splanchnic pathways did not reduce VMRs following acute noxious
colorectal distension, but were abolished by rectal nerve lesions.
A previous study by Robinson et al. (2004) injected fast blue
into the descending colon of mice and also identified two peaks of
DRG distribution, one peak at the level of T7 and the other at L3.
It was stated that fast blue was injected into the descending colon
via laparotomy, but it is not exactly clear how far in millimeters
the fast blue was injected into the colon from the anus. One of the
major findings of the study by Robinson et al. (2004) was that spinal
afferents innervating the descending colon were largely located
in thoracolumbar segments (T11-L1) segments. This appears to
contrast with our findings, since we found predominantly thoracic
labeling of DRG neurons when DiI was injected into the mid to
proximal region of colon, at a distance of 30–75 mm from the anus
(measured in unstretched preparations). We used the same strain
and similar age of mouse as in the study of Robinson et al. (2004),
but different neuronal tracers were used. It is possible, but seems
difficult to reconcile how different tracers would reveal different
distributions of DRG labeling.
In a study by Tan et al. (2008) cholera toxin B was injected into
two sites in the mouse descending colon, ∼ 20 mm from the anus.
In that study, it was reported that DRGs innervating this region
were primarily located in T13-L1 and L4-S1. The results of Tan et al.
(2008) are similar to our findings. Also, a study by Christianson
et al. (2007) injected cholera toxin B into the mouse descending
colon, at distances that were 10 and 20 mm from the anus. These
understand the origin and types of afferents important for signaling
nociceptive stimuli to the central nervous system. In this study, we
developed a novel preparation in anesthetized mice in which it was
possible to visualize the major spinal afferent pathways that com-
municate between the colorectum and spinal cord, whilst applying
noxious distension stimuli to the colorectum. This allowed us to
systematically lesion each of the major afferent pathways to une-
quivocally determine which pathway(s) were required for signaling
noxious distension stimuli from the colorectum.
Using this novel preparation, the major finding of the cur-
rent study has shown the rectal/pelvic nerve pathway is essential
for the detection and transmission of VMRs elicited by acute
noxious distension of the terminal 15 mm of colorectum. This
conclusion was based on the finding that lesions applied to the
rectal nerves always abolished VMRs to colorectal distension,
but lesions to the lumbar colonic or hypogastric nerves had no
measurable effect.
sensory Pathway Involved In detectIon of noxIous colorectal
dIstensIon
Identification of the nociceptive pathways from the terminal region
of the GI tract has been a subject of much speculation (Brierley et
al., 2009; Hughes et al., 2009; Feng et al., 2010). In our study, when
DiI was injected into the distal colon, at a distance of 9–15 mm
from the anus (measured in unstretched preparations), the sensory
neurons retrogradely labeled were found at the level of the lum-
bosacral spinal cord (peak at L6-S1). This suggests that distension
of the distal colon, at the transition zone between the rectal nerves
and LCN utilizes sensory neurons primarily in the lumbosacral
Figure 3 | Distribution of retrogradely labeled Drg neurons following
injection of Dii into the mouse distal colon. (A) Following DiI injections
made 9–15 mm from the anal sphincter (measured from unstretched
colorectum preparations), the distribution of retrogradely labeled DRG
neurons were identified. The majority of labeled neurons were identified in
the lumbosacral region of spinal cord. Primary labeling was identified
at levels of L6 and S1. (B) Fluorescence micrograph showing labeled
neurons at L6.

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Kyloh et al. Pain pathway activated by colorectal distension
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marily located in the lumbosacral region, with only a small propor-
tion of neurons labeled in thoracolumbar DRGs.
During our study, we also had an opportunity to assess function-
ally the relative contributions of thoracolumbar and sacral sensory
pathways by severing acutely the major nerve trunks, selectively. As
mentioned above, acute lesions to either, or both of the hypogastric
nerves or the LCN had no detectable effect on the amplitude of
VMRs. However, cutting the rectal nerves consistently abolished
the response regardless of whether lumbar colonic or hypogastric
nerves were severed. This strongly supports our retrograde tracing
studies that show sacral afferents, traveling via rectal and pelvic
nerves to the distal bowel, are the primary pathway involved in
detecting acute noxious distension of the terminal rectum (∼15 mm
of unstretched rectum). This conclusion is further substantiated
by the observation that VMRs could be reliably elicited following
electrical stimulation of the rectal nerves, but not by the lumbar
colonic or hypogastric nerves. Our conclusions that the VMRs elic-
ited by acute noxious colorectal distension is transmitted via the
rectal/pelvic afferents is remarkably similar to the findings reported
by Nagabukuro and Berkley (2007), who distended the vagina to
elicit VMRs. They also reported that lesions applied to the hypogas-
tric nerves did not reduced VMRs to vaginal distension, but were
abolished by pelvic nerve lesions.
densIty of drg neurons InnervatIng colorectuM Is hIgher
than that those of MId–ProxIMal colon
One of the major findings of this study was the distinct dif-
ferences in density of DRG neurons labeled from DiI injec-
tions made in the colorectum compared with those made into
mid–proximal colon. Following colorectal injections of DiI the
number of labeled neurons was approximately 20 times higher
per DRG section, compared with DiI injections into the mid–
proximal colon, whilst maintaining all other parameters constant
(volume of dye injected, number of sites injected, time allowed
for uptake, etc). On average, there were typically only about
one to two labeled neurons per section from mid– proximal
colon injections. The reason why the sensory innervation of
the colorectum is considerably greater than the mid–proximal
colon is not clear, but may be related to additional afferent nerve
pathways recruited during the defecation reflex. The greater
density of spinal afferents innervating the rectum conceptually
seems similar to the increased density of intestinofugal neu-
rons which also increases closer to the rectum (Messenger and
Furness, 1992).
conclusIon
The results of this study show that the visceral pain pathway
activated by acute noxious distension of the terminal 15 mm
of colorectum is mediated via the rectal/pelvic spinal afferents,
whose cell bodies lie primarily in the lumbosacral region of
the spinal cord. No evidence was found to suggest that lum-
bar splanchnic afferents are required for the detection and
transmission of noxious mechanical stimuli from this region
of colorectum.
acknowledgMents
We wish to acknowledge the financial support provided by the
National Health and Medical Research Council (NH&MRC) of
Australia to grant numbers 535033 and 535034.
Figure 4 | Distribution of retrogradely labeled Drg neurons following injection of Dii into the mid to proximal colon. (A) DiI injected into the mouse colon
30–75 mm from the anus (measured in unstretched colon preparations, N = 5). DiI labeled neurons were primarily identified between T5 and L2, with peak labeling
between T11-L1. Only one to four neurons on average were labeled in any DRG section. (B) Shows a fluorescence micrograph of labeled neurons at T12.

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www.frontiersin.org February 2011 | Volume 5 | Article 16 | 7
Kyloh et al. Pain pathway activated by colorectal distension
Zagorodnyuk, V. P., Kyloh, M., Nicholas,
S., Peiris, H., Brookes, S. J., Chen, B.
N., and Spencer, N. J. (2011). Selective
loss of visceral pain in the rectum of an
endothelin-3 deficient mouse model
of Hirschsprung’s disease. J. Physiol.
(in press).
Conflict of Interest Statement: The
authors declare that the research was con-
ducted in the absence of any commercial
or financial relationships that could be
construed as a potential conflict of interest.
Received: 26 November 2010; accepted:
27 January 2011; published online: 22
February 2011.
Citation: Kyloh M, Nicholas S, Zagorodnyuk
VP, Brookes SJ and Spencer NJ (2011)
Identification of the visceral pain pathway
activated by noxious colorectal distension in
mice. Front. Neurosci. 5:16. doi: 10.3389/
fnins.2011.00016
This article was submitted to Frontiers in
Autonomic Neuroscience, a specialty of
Frontiers in Neuroscience.
Copyright © 2011 Kyloh, Nicholas,
Zagorodnyuk, Brookes and Spencer. This
is an open-access article subject to a non-
exclusive license between the authors and
Frontiers Media SA, which permits use, dis-
tribution and reproduction in other forums,
provided the original authors and source are
credited and other Frontiers conditions are
complied with.
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