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A STUDY OF PRO- AND ANTI-NOCICEPTIVE FACTORS IN A MODEL OF COLITISASSOCIATED VISCERAL PAIN
by
Jessica Rose Benson
A thesis submitted to the Physiology Graduate Program in the Department of
Biomedical and Molecular Sciences in conformity with the requirements for the degree
Master of Science
Queen’s University
Kingston, Ontario, Canada
(August, 2012)
Copyright © Jessica Rose Benson, 2012
Abstract
Chronic abdominal pain is a major cause of patient morbidity in inflammatory
bowel diseases (IBD). A balance of pro- and anti-nociceptive factors regulating colonic
dorsal root ganglion (DRG) neurons, which synapse onto second order dorsal horn
neurons, are known to regulate chronic pain but the mechanisms are poorly understood.
This thesis examined whether neuroanatomical remodeling of DRG central nerve
terminals underlies pro-nociceptive signaling and whether subsets of immune cells
source the anti-nociceptive factor, β-endorphin.
To examine pro-nociceptive mechanisms, acute and chronic dextran sulfate
sodium (DSS) mouse models of colitis were established and substance P (SP; marker
of nociceptor terminals) immunohistochemistry used to investigate changes in
immunoreactivity of DRG terminals in the thoracic dorsal horn (segments T9-T13). SP
immunoreactivity was increased in the dorsal horn (4 fold; P < 0.001) and central canal
(P < 0.001) following chronic colitis. In contrast, SP immunoreactivity was unchanged in
acute colitis. However, five weeks later SP immunoreactivity was increased both in the
dorsal horn (4 fold; P < 0.01) and central canal (P < 0.001). In the cervical spinal cord,
SP immunoreactivity was not increased following colitis, suggesting that changes seen
in the thoracic level were specific to signaling from colonic DRG neurons.
Immunoreactivity for the SP NK1 receptor on second order neurons was also examined
and a significant increase in immunoreactivity was observed on post-synaptic second
order cell bodies following chronic DSS. This could provide an additional mechanism for
enhanced SP neurotransmission centrally.
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The source of the anti-nociceptive mediator, β-endorphin, during chronic DSS
colitis was investigated using magnetic cell sorting and flow cytometry. The number of βendorphin expressing CD4+ (2.4 fold; P < 0.05) and CD11b+ (2.6 fold; P < 0.05) cells in
mice increased following chronic colitis.
These findings suggest that during colitis there is a time-dependent increase of
SP immunoreactivity in thoracic DRG central terminals, which could play a role in pronociceptive signaling in chronic inflammation. These actions may be balanced by antinociceptive factors such as β-endorphin which are found in subsets of immune cells.
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Co-Authorship
Drs. Alan Lomax and Stephen Vanner provided the conceptual framework for the
ideas on which this thesis was based, as well as feedback on experimental design and
results. Dr. Lomax also provided me with training in immunohistochemistry, data
collection, and statistical analyses. In addition Dr. Vanner has helped me to develop
critical writing skills.
Dr. Ian Spreadbury, a research associate in our laboratory, assisted me in
weighing and caring for mice undergoing dextran sulfate sodium colitis. Ms. Shadia
Neshat, our laboratory technician, assisted me in adopting the protocol used for lamina
propria cell dissociations and provided me with training for this technique. Ms. Iva
Kosatka and Margaret O’Reilly, our laboratory technician and research associate,
respectively, performed transcardial perfusions on mice with colitis and control mice,
and performed thoracic and cervical spinal cord tissue dissections. Additionally, Dr.
Vandana Gambhir, a post-doctoral fellow of the Vanner laboratory, provided me with
training in magnetic cell sorting and flow cytometry.
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Acknowledgements
First and foremost, I would like to thank my co-supervisors, Dr. Alan Lomax and
Dr. Stephen Vanner, for their unwavering support and encouragement throughout the
duration of my graduate studies. I have been lucky enough to experience this research
project under the guidance of not one, but two, exceptional supervisors. I would
especially like to thank my co-supervisors for their unmatched wisdom and for believing
in me throughout this entire process.
I would also like to thank members of the Lomax and Vanner laboratories for
being great colleagues that have brought joy and laughter into each of my work days.
Iva Kosatka, Shadia Neshat, and Margaret O’Reilly have provided me with excellent
assistance, encouragement, and friendship throughout my research project. Dr. Ian
Spreadbury has been a profound friend and colleague who has believed in me from day
one. Thank you, Ian, for the multitude of motivational speeches and for your quirky
sense of humour. In addition, I would like to thank the students of the Lomax lab who
have contributed greatly to my experiences in GIDRU. Thank you to Mark Lukewich,
Andrea Cervi, Lauren Steinhart, Simrin Nagpal, and Derek Moynes for your friendships
and assistance. Without the support from these great individuals, I would not be where I
am today.
Lastly but certainly not least, I would like to thank my family and friends for their
incredible support. To my family, I additionally thank you greatly for your patience! When
times got tough, I knew I could always count on you to be there. Mom, thank you for
being my rock throughout this entire journey; you inspire me everyday to work hard and
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to face obstacles head on. Krista, thank you for being the best sister in the world; I can
always count on you to be there to hold me together when I feel close to falling apart.
Dad, thank you for your endless support and love of my cat; I know she will always be in
good hands when you are around! Jeff, even though you are miles away, I know that
you have only wished the best for me throughout my studies, and I thank you for being
my support away from home. You are the best brother in the world! To my friends, I
would like to say that you are all such wonderful people and I am truly blessed to have
you in my life. To my teammates I say thank you for a fantastic two years! It has been an
honour playing with all of you, and your support both on the field and off has motivated
me every day to reach for new goals.
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Table of Contents
Abstract............................................................................................................................ ii
Co-Authorship..................................................................................................................iv
Acknowledgements.......................................................................................................... v
Chapter 1 Introduction..................................................................................................... 1
1.1 Inflammatory Bowel Diseases.............................................................................. 1
1.1.1 Crohn’s Disease and Ulcerative Colitis......................................................... 1
1.1.2 Major Symptoms of IBD................................................................................ 2
1.2 Anatomical Pathways of Pain Neurotransmission From the Gut.......................... 2
1.2.1 The Nociceptor............................................................................................. 4
1.2.2 Peripheral Sensitization of the Nociceptor.................................................... 7
1.2.3 Central Ascending Sensory Afferent Pathways in Pain Transmission.........13
1.2.4 Sensitization of Ascending Pathways..........................................................14
1.2.5 Nociceptors: Peripheral Versus Central Studies..........................................15
1.3 Inflammation-Dependent Anti-Nociception of Pain Pathways.............................17
1.3.1 Inflammatory Mediators That Promote Analgesia........................................17
1.3.2 β-Endorphin Regulation of Nociception.......................................................20
1.4 Hypothesis...........................................................................................................23
1.4.1 Objectives Part 1: Pro-Nociception..............................................................24
1.4.2 Objectives Part 2: Anti-Nociception.............................................................24
Chapter 2 Methods.........................................................................................................25
2.1 Animal Model.......................................................................................................25
2.2 Introduction of Colitis...........................................................................................25
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2.3 Immunohistochemistry.........................................................................................26
2.3.1 Tissue Cryosections....................................................................................26
2.3.2 Quantification of Immunofluorescence........................................................28
2.4 Flow Cytometry....................................................................................................29
2.4.1 Lamina Propria Cell Isolation.......................................................................29
2.4.2 Magnetic Cell Sorting...................................................................................30
2.4.3 Endogenous Opioid Labeling.......................................................................31
2.4.4 Flow Cytometry............................................................................................31
2.5 Statistical Analysis...............................................................................................32
Chapter 3 Results...........................................................................................................33
3.1 Development of Colitis.........................................................................................33
3.2 Controls...............................................................................................................33
3.3 Results Part 1: Pro-Nociception...........................................................................38
3.3.1 DSS-induced colitis increases DRG SP innervation in the dorsal horn of the
thoracic spinal cord.........................................................................................................38
3.3.2 Colitis increases SP immunoreactivity in the central canal of the thoracic
spinal cord......................................................................................................................40
3.3.3 SP immunoreactivity is decreased in the cervical spinal cord segments C2C4 following chronic DSS colitis.....................................................................................40
3.3.4 Effect of chronic DSS colitis on SP immunoreactivity in the colon..............42
3.3.5 NK1R is increased following chronic DSS on post-synaptic cells located in
the dorsal horn of the thoracic spinal cord......................................................................42
3.4 Results Part 2: Anti-Nociception..........................................................................46
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3.4.1 Chronic DSS colitis increases expression of the endogenous opioid βendorphin in lamina propria isolated CD4+ and CD11b+ immune cells.........................46
Chapter 4 Discussion......................................................................................................49
4.1 Time-dependent increase of SP immunoreactivity in the thoracic spinal cord
segments T9-T13............................................................................................................49
4.1.1 Evidence for neuroanatomical remodeling..................................................52
4.2 SP immunoreactivity in colonic tissue..................................................................54
4.3 SP immunoreactivity in the cervical spinal cord...................................................55
4.4 NK1R immunoreactivity is increased in the thoracic spinal cord following chronic
colitis...............................................................................................................................56
4.5 Future studies for pro-nociceptive mechanisms..................................................58
4.6 Sources of β-endorphin in chronic DSS colitis.....................................................59
4.7 Future studies for anti-nociceptive mechanisms..................................................61
4.8 Concluding Remarks...........................................................................................63
References.....................................................................................................................65
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List of Figures
Figure 1. Anatomy of colonic pain pathways................................................................3
Figure 2. Peripheral mediators of inflammation........................................................... 8
Figure 3. Incubation of naive DRG neurons in supernatant from colons of chronic DSStreated mice decreases neuronal excitability..............................................................19
Figure 4. Weight change in acute DSS-treated mice................................................. 34
Figure 5. Weight change in post-acute DSS-treated mice......................................... 35
Figure 6. Weight change in chronic DSS-treated mice.............................................. 36
Figure 7. Weight change in post-chronic DSS-treated mice...................................... 37
Figure 8. Substance P (SP) immunoreactivity in the dorsal horn of the thoracic spinal
cord segments T9-T13 is increased following acute and chronic DSS colitis............ 39
Figure 9. Substance P (SP) immunoreactivity in the central canal of the thoracic spinal
cord segments T9-T13 is increased following acute, chronic, and post-chronic DSS
colitis.......................................................................................................................... 41
Figure 10. Substance P (SP) immunoreactivity in the dorsal horn of the cervical spinal
cord segments C2-C4 is decreased following chronic colitis..................................... 43
Figure 11. Substance P (SP) immunoreactivity in the mucosa and muscularis externae
of the distal colon is unchanged following chronic colitis........................................... 44
Figure 12. Neurokin-1 Receptor (NK1R) immunoreactivity in the dorsal horn of the
thoracic spinal cord segments T9-T13 is increased following chronic colitis............. 45
Figure 13. β-endorphin is positively expressed in CD4+ and CD11b+ cells and is
increased following chronic DSS colitis...................................................................... 48
Figure 14. Potential mechanisms for increased SP innervation of the spinal cord.... 53
Figure 15. Nociception is determined by a balance of pro- and anti-nociceptive
mediators................................................................................................................... 64
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List of Abbreviations
ASIC
Acid Sensitive Ion Channels
BDNF
Brain-Derived Neurotrophic Factor
BSA
Bovine Serum Albumin
CB1
Cannabinoid Receptor 1
CCI
Chronic Constriction Injury
CD
Crohn’s Disease
CGRP
Calcitonin-Gene Related Peptide
CRD
Colorectal Distensions
CRF
Corticotropin-Releasing Factor
CTB
Cholera Toxin B Subunit
DAPI
4',6-Diamidino-2-Phenylindole
DSS
Dextran Sulfate Sodium
DTT
Dithiothreitol
DOR
δ-Opioid Receptor
DRG
Dorsal Root Ganglia
EDTA
Ethylenediaminetetraacetic Acid
ELISA
Enzyme-Linked Immunosorbent Assay
FAE
Follicle-Associating Epithelium
FBS
Fetal Bovine Serum
FCA
Freund’s Complete Adjuvent
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GABA
γ-Aminobutyric Acid
GAP
Growth Associated Protein
GI
Gastrointestinal
IB4
Isolectin B4
IBD
Inflammatory Bowel Diseases
IBS
Irritable Bowel Syndrome
ICAM1
Intercellular Adhesion Molecule 1
IFN-γ
Interferon-γ
K2P
Two-Pore Domain Potassium
KOR
κ-Opioid Receptor
LPS
Lipopolysaccharide
MACS
Magnetic Activated Cell Sorting
MAPK
Mitogen-Activated Protein Kinase
MOR
µ-Opioid Receptor
MPO
Myeloperoxidase
NGF
Nerve Growth Factor
NK1R
Neurokinin Receptor 1
NOFQ
Nociceptin/Orphanin FQ
ORL1
Opioid Receptor-Like Receptor 1
PAR2
Protease-Activated Receptor 2
PBS
Phosphate Buffered Saline
PCR
Polymerase Chain Reaction
PE
Phycoerythrin
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pERK
Phosphorylated Extracellular Signal-Regulated Kinases
PFA
Paraformaldehyde
PGP
Protein Gene Product 9.5
PP
Peyer’s Patches
PSDC
Post-Synaptic Dorsal Column
ROI
Regions of Interest
RPMI
Roswell Park Memorial Institute
RTK
Receptor Tyrosine Kinase
SCID
Severe Combined Immundeficient
SEM
Standard Error of the Mean
SP
Substance P
TNBS
2,4,6-Trinitrobenze Sulfonic Acid
TNF
Tumour Necrosis Factor
trkA
Tropomyosin-Receptor-Kinase A
TRPV
Transient Receptor Potential Vanilloid 1
TTX
Tetrodotoxin
UC
Ulcerative Colitis
VGSC
Voltage-Gated Sodium Channels
WT
Wild Type
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Chapter 1
Introduction
1.1 Inflammatory Bowel Diseases
IBD defines a group of idiopathic diseases that are characterized by chronic
inflammation of the GI tract. The pathogenesis of IBD is unknown but there is strong
evidence that both environmental and genetic factors act synergistically to affect
disease onset and severity (Podolsky 2002). Canada has one of the highest
prevalences of IBD in the world; IBD affects over 200,000 individuals and in 2008
medical costs alone were approximately 1.8 billion dollars (CCFC 2008; http://
www.ccfc.ca/site/c.ajIRK4NLLhJ0E/b.6431205/k.884D/
The_Burden_of_IBD_in_Canada.htm). These disorders also have a tremendous social
cost as affected individuals are often young at the time of onset (Longobardi et al. 2003;
Szigethy et al. 2010).
1.1.1 Crohn’s Disease and Ulcerative Colitis
Crohn’s disease (CD) and ulcerative colitis (UC) are the two most common types
of IBD and can often be differentiated by the distribution and nature of the inflammation
in the GI tract (Clarke et al. 2008). CD can affect the GI tract from the oral cavity to the
anus, however the terminal ileum is the most commonly affected region (Ament 1975).
In addition, CD is a transmural disease meaning that it can involve the mucosa,
submucosa, muscle layers, and serosa. On the other hand, UC is restricted to the colon
and the inflammation always extends continuously from the anus for some distance
proximally in the colon (Ament 1975). Unlike CD, UC only affects the mucosa and
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submucosa within the colon. All forms of IBD may lead to dysregulation of the bowel
and the onset of chronic, debilitating symptoms (Clarke et al. 2008).
1.1.2 Major Symptoms of IBD
Major symptoms of IBD include weight loss, diarrhea, rectal bleeding, and
abdominal pain. In particular, chronic abdominal pain is a leading cause of morbidity in
patients with IBD (Pizzi et al. 2006) and is a difficult symptom to treat due to its chronic
nature and because the pathogenesis of IBD and resulting pain are poorly understood.
Thus, investigation of the mechanisms underlying chronic abdominal pain is of critical
importance to improve the quality of life in patients suffering from IBD. Modulation of the
neural pain pathway causing exaggerated nociceptive signaling from the inflamed
tissues is a critical component to the development of chronic abdominal pain, and is
explained in further detail below (Beyak et al. 2004; Jones et al. 2005; Szallasi et al.
2007; La and Gebhart, 2011). As a result, the major focus of my research was to study
how inflammatory modulation of the pain pathway is involved in the onset of chronic
abdominal pain in IBD. Using experimental mouse models of colitis, I specifically
address how sensory neural circuits innervating the colon are influenced by a balance
of both pro-nociceptive and anti-nociceptive mechanisms.
1.2 Anatomical Pathways of Pain Neurotransmission From the Gut
An anatomical overview of the pain pathway involved in IBD is depicted in Figure
1. In general, the detection of painful stimuli in the colon is mediated by a subset of
DRG sensory neurons called nociceptors (Basbaum et al. 2009).
2
Cingulate cortex
Somatosensory
cortex
Insular cortex
Thalamus
Amygdala
PAG
PB
RVM
Brain Stem
FIGURE 1. Anatomy of Colonic Pain Pathways.
Colonic-innervating nociceptors relay noxious information to projection neurons within the dorsal horn of
the spinal cord. From here, projection neurons transmit information to the somatosensory cortex via the
thalamus to provide information about the location and intensity of the noxious stimulus. Projection
neurons also engage the cingulate and insular cortices via connections in the brainstem and amygdala
to contribute to the emotional component of the pain experience. Lastly, ascending information also
accesses neurons of the rostral ventral medulla and midbrain periaqueductal gray to engage
descending feedback systems that regulate output from the spinal cord. Noxious neuropeptides
substance P and calcitonin-gene related peptide are released centrally to transmit noxious information
from the colon to ascending pain pathways. Abbreviations: PAG periaqueductal gray; PB parabrachial
nucleus; RVM rostroventral medulla.
* SP and CGRP neuropeptides also have local, “effector” functions in the peripheral terminals that upon
release, contribute to neurogenic inflammation through mechanisms such as increased vascular
permeability. Modified from Basbaum et al. 2009.
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These pseudo-unipolar neurons have both peripheral and central afferent axonal
terminals. The central terminals synapse onto second order neurons located in the
dorsal horn of the spinal cord (Light and Perl, 1979; Sugiura et al. 1986). These neurons
project to multiple central sensory nuclei (see Figure 1) where location, intensity, and
emotional components are perceived (Chapman et al. 1985; Palecek et al. 2004; Ren et
al. 2009; Wouters et al. 2012). Other projections also access neurons of the rostral
ventral medulla and midbrain periacqueductal gray to engage descending feedback
systems and regulate output from the spinal cord (Wouters et al. 2012).
Changes in the sensitivity of the pain pathway are believed to be an important
component underlying enhanced nociceptive signaling in IBD (Beyak et al. 2004; Jones
et al. 2005; Ibeakanma and Vanner 2010). Thus, the following sections will provide a
thorough overview of the anatomy of colonic pain pathways and discuss how each
component can become sensitized as a result of inflammation.
1.2.1 The Nociceptor
Nociceptors are distinguished by high activation thresholds, a small cell diameter,
and the presence of multiple ion channels that are implicated in nociceptor sensitization
(Woolf and Ma, 2007). Such channels include the transient receptor potential vanilloid
(TRPV) receptor 1 (Caterina et al. 1997), the protease-activated receptor (PAR)-2 (Gill
et al. 1998; Steinhoff et al. 2000), and the tetrodotoxin (TTX)-resistant voltage-gated
sodium channel (VGSC) Nav1.8 (Renganathan et al. 2001; Blair and Bean, 2002;
Zimmermann et al. 2007). In addition, nociceptors can be distinguished by the
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neurotransmitters and neuropeptides they produce, such as glutamate, SP and CGRP
(Tuchscherer and Seybold, 1989).
In the colon, nociceptive signals are relayed by neurons with cell bodies located
in the DRG T9-T13 and L6-S1 (Robinson et al. 2004). Proteins synthesized by the
nucleus of the nociceptor neurons can be transported to both peripheral and central
nerve endings, giving nociceptors the capability to have dynamic regulatory roles at
both peripheral and central interfaces (Basbaum et al. 2009). There are different
subtypes of nociceptors, characterized by diameter size, action potential conductance
velocity, and neuropeptide composition (Bennett et al. 1996; Molliver et al. 1997;
Almeida et al. 2004). Discussed in further detail below are the two major subtypes of
nociceptors: Aδ fibers and C fibers.
The Aδ fiber subtype nociceptors are thinly myelinated (~2-6 µm in diameter) and
respond to mechanical, thermal, and chemical stimuli (Sengupta and Gebhart 1994;
Caterina et al. 1999; Almeida et al. 2004; Kobayashi et al. 2005). With a conduction
velocity of approximately 2-30 m/s, Aδ fibers are considered to be fast-response
neurons (Almeida et al. 2004). Aδ fibers are divided into subclasses with different
activation thresholds (Meyer et al. 2008). Whereas both subclasses of Aδ fibers are
responsive to mechanical and thermal stimuli, one subclass of Aδ fibers has a high
thermal threshold of activation and the other contains a high mechanical threshold of
activation. All Aδ fibers have central terminals that synapse in laminae I and V of the
dorsal horn (Light and Perl 1979).
The C fiber subtype of nociceptors is comprised of unmyelinated neurons with
small diameter axons (~ 0.4-1.2 µm) and transmit at a slower conduction velocity of < 2
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m/s (Almeida et al. 2004). Like Aδ fibers, C fibers are also divided into subclasses and
are responsive to mechanical, thermal, and chemical stimuli (Bessou and Perl 1969).
Electrophysiological recordings from colonic afferent peripheral endings show that these
fibers have high- or low-mechanosensory thresholds (Brierley et al. 2004; Brierley et al.
2005). In addition, C fibers are distinguished as peptidergic or non-peptidergic. The
peptidergic population of C fibers positively express the neuropeptides SP and CGRP,
and are found in abundance in colon-innervating DRG (Robinson et al. 2004; Woolf and
Ma, 2007). Peptidergic C fibers can also be identified by their positive expression of the
tropomyosin-receptor-kinase A (trkA) neurotrophin receptor (Averill et al. 1995). Nonpeptidergic C fibers are identified by the uptake of isolectin B4 (IB4) and expression of
the neurotrophin receptor c-Ret (Molliver et al. 1997; Bennett et al. 1998). Although nonpeptidergic C fibers are present in colon-innervating DRG, the peptidergic population of
C fibers comprise the majority of colon-innervating nociceptive neurons (Robinson et al.
2004). The central terminals of C fibers mainly synapse in the superficial dorsal horn of
the spinal cord, in laminae I and II (Sugiura et al. 1986).
Given the role of SP in colonic nociceptive neurons, SP immunoreactivity
represents a reliable marker of nociceptive DRG neurons. SP belongs to the tachykinin
family of neuropeptides that bind to the neurokinin receptors (NK1R, NK2R, NK3R). SP
has greatest binding affinity for NK1R (Helke et al. 1990), and any changes in SP and
NK1R immunoreactivity in the dorsal horn of the spinal cord may provide important
information regarding the plasticity of pre-synaptic nociceptor nerve terminals and postsynaptic second order neurons, respectively.
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1.2.2 Peripheral Sensitization of the Nociceptor
Peripheral sensitization is a neurophysiological change in which the peripheral
terminals of nociceptors become more sensitive to a variety of sensory stimuli
(Basbaum et al. 2009). Sensitization of the nociceptor can occur by the up-regulation of
ion channel expression, augmented ion channel current conductance, and/or the upregulation of receptor expression on peripheral terminals (Beyak et al. 2004; Akbar et al.
2010). These neurophysiological changes result in a decreased threshold for action
potential discharge and/or increased action potential discharge in response to a given
stimulus. In IBD, peripheral sensitization arises from various inflammatory mediators
acting on peripheral terminals of sensory afferent neurons. A summary of the ion
channel targets and sensitization mediators can be found in Figure 2. Discussed below
are examples of some of the better understood peripheral mechanisms responsible for
inflammatory sensitization of nociceptors.
TRPV1 non-selective cation channels play an important role in the development
of peripheral sensitization of nociceptors in the colon. Primarily, TRPV1 channels are
present on C fiber nociceptors which are the most frequently occurring nociceptor in the
colon (Robinson et al. 2004; Adam et al. 2006). TRPV1 channels are activated by
capsaicin, protons, and heat (Ross 2003). Activation of these channels allows for cation
influx, which in turn contributes to depolarization of nociceptors. Increased
depolarization through augmented ion current flow within the TRPV1 channel pore and
through increased expression on peripheral terminals following inflammation can in turn
decrease the activation threshold of the nociceptor and contribute to the generation of
action potentials, or nociceptor excitability.
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Tissue damage
Mast cells
Platelets
Macrophage
Immune cells
Adenosine
Histamine
H
+
ATP
Bradykinin PGE2
IL-6 NGF IL-1â TNF-á
Substance P
CGRP
ASIC/P2X
K2P
RTK
GPCR
TRP
FIGURE 2. Peripheral Mediators of Inflammation
Tissue damage leads to the release of inflammatory mediators from activated nociceptors and from nonneural cells that reside within or infiltrate into the injured area. Such mediators include mast cells,
basophils, platelets, macrophages, neutrophils, endothelial cells, keratinocytes, and fibroblasts.
Inflammatory mediators in turn release numerous signaling molecules including serotonin, histamine,
glutamate, ATP, adenosine, substance P, calcitonin-gene related peptide (CGRP), bradykinin, eicosinoids
prostaglandins, thromboxanes, leukotrienes, endocannabinoids, nerve growth factor (NGF), tumor
necrosis factor á (TNF-á), interleukin 1â (IL-1â), extracellular proteases, and protons. These factors act
directly on the nociceptor by binding to one or more cell surface receptors, including G protein coupled
receptors (GPCR), TRP channels, Acid-sensitive ion channels (ASIC), two-pore potassium channels
(K2P), and receptor tyrosine kinases (RTK), as depicted on the peripheral nociceptor terminal. Modified
from Basbaum et al. 2009.
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Studies have shown that mice lacking the TRPV1 channel have lower sensitivity to
intestinal distension (Jones et al. 2005). Furthermore, the hypersensitivity that develops
in response to the presence of inflammatory mediators in wild-type mice is abolished in
TRPV1-deficient mice, and pharmacological inhibition of TRPV1 blocks the
development of hyperalgesia during colitis (Jones et al. 2005; Szallasi et al. 2007). Thus
these findings implicate TRPV1 as an important contributor to the development of
visceral hypersensitivity. Clinical research has shown that TRPV1 immunoreactive
axons increase ~4 fold in rectosigmoid biopsies of patients with quiescent IBD (both
symptomatic and asymptomatic), and may contribute to the pathophysiology of ongoing
pain (Akbar et al. 2010). Overall, TRPV1 has a well characterized role in the
development of visceral hypersensitivity.
PAR-2 is a G-protein coupled receptor expressed by nociceptive neurons
(Steinhoff et al. 2000) and is activated during inflammation by proteases released from
mast cells, the intestinal lumen, and the circulation. Inflammatory agents including mast
cell tryptase, trypsins, coagulation factors VIIa and Xa, and kallikreins, cleave and
activate PAR-2 (Kawabata et al. 2006). Activated PAR-2 stimulates the release of SP
and CGRP from nerve terminals in peripheral tissue (Nguyen et al. 2003). This is of
particular importance as peripheral release of SP increases vascular permeability
(Lordal et al. 1996). This would allow circulating cytokines and pro-inflammatory
mediators to infiltrate the inflamed region of the GI tract more easily, giving peripheral
SP a pro-inflammatory role. In turn, pro-inflammatory mediators can sensitize DRG
nociceptors (see below). Thus, PAR-2 activated release of SP from nociceptors and
subsequent development of neurogenic inflammation is an indirect contribution to
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peripheral sensitization. In addition, PAR-2 have been implicated in the induction of
hyperalgesia to colonic distension following TNBS colitis (Cattaruzza et al. 2011).
However, the mechanisms underlying this hyperalgesia are currently unknown. Thus far,
it is evident that PAR2 plays an important role in nociceptor sensitization through
indirect modulation of peripheral sensitization, and through regulation of mechanical
sensitivity.
The TTX-resistant VGSC Nav1.8 and Nav1.9 are also expressed by C fiber
nociceptors and contribute to the control of membrane excitability. Activated VGSC
cause an influx of Na+ into the cell that underlies action potential generation in DRG
neurons (Leo et al. 2010). Na+ currents generated by these channels are augmented by
inflammation; The Nav1.8 current has been shown to significantly increase following
colitis in CGRP- and trkA-positive DRG neurons (Beyak et al. 2004). In addition, colitis
leads to increased expression of the Nav1.8 channel in colonic DRG neurons. The
Nav1.9 channel has also shown evidence for a role in the development of hyperalgesia
following both inflammatory and neuropathic pain (Leo et al. 2010). Inflammatory
modulation of VGSC is thus an important contributor to nociceptor sensitization in IBD.
Acid sensitive ion channels (ASIC) subunits 1-3 are expressed in DRG neurons
(Yiangou et al. 2001). These channels are proton-gated, voltage-independent, and
mainly permeable to Na+ ions (Waldmann et al. 1997; Deval et al. 2010). ASIC currents
activate transiently upon extracellular acidification and lead to increased intracellular
release of Ca+ (Xiong et al. 2004; Yermolaieva et al. 2004). Colonic inflammation leads
to the release of protons from damaged tissue. Thus, exposure of these channels to a
lowered pH following colonic inflammation may be important in the regulation of DRG
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neuronal cell excitability, and may play a role in the generation of peripheral sensitivity
in IBD. Particularly, ASIC currents may be involved with altered mechanosensory
function following inflammation, as it has been shown that mice lacking ASIC1a, ASIC2,
and ASIC3 subunits exhibit altered colonic mechanotransduction (Page et al. 2005).
Two-pore domain K+ (K2P) channels are voltage-independent K+ channels that
play an important role in regulating cell excitability by setting and shaping the resting
membrane potential and action potential (La and Gebhart, 2011). K2P channels have
outward K+ currents, where K+ ions leave the cell to regulate membrane potential.
Decreased conductance of these channels would in turn increase cell depolarization
and the likelihood of action potential generation, and contribute to neuronal cell
excitability. There are six subfamilies of K2P channels described to date including the
TREK subfamily (Kim 2005). In mouse DRG, the majority of neurons positively express
at least one of TREK-1, TREK-2, or TRAAK mRNA, which are all members of the TREK
subfamily of K2P channels (La and Gebhart, 2011). These TREK channels are activated
by membrane stretch, heat, intracellular acidosis, alkalosis, or lipids suggesting these
channels are involved in mechanosensation, thermosensation, and chemosensation
(Maingret et al. 1999; Bang et al. 2000; Kang et al. 2005). With respect to colonic
inflammation, studies have implicated these channels as contributors to increased
sensitivity of DRG neurons (La and Gebhart, 2011). Specifically following colonic
inflammation, decreased TREK-1 mRNA expression, decreased responsiveness of
TREK-2-like channels to membrane stretch, and decreased whole cell outward current
to osmotic stretch were observed in mouse colon DRG neurons (La and Gebhart, 2011).
The decreases observed suggest that K2P channels contribute to increased colon
11
mechanical sensitivity following colitis through down-regulated expression and
conductance.
The receptor tyrosine kinase (RTK) receptors are high-affinity cell surface
receptors for many growth factors, cytokines, and hormones. Nociceptors positively
express the Trk subfamily of RTK receptors that bind neurotrophins such as nerve
growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (Martin-Zanca et al.
1986; McMahon et al. 1994; Averill et al. 1995; Phillips and Armanini, 1996). When
bound, Trk receptors lead to the activation of multiple signaling cascade pathways.
These cascades lead to the regulation of transcriptional activity, and may be important
in the development of nociceptor sensitization. In fact, multiple studies have shown that
NGF and BDNF play a role in the development of peripheral sensitization (Groth and
Aanonsen, 2002; Delafoy et al. 2003; Delafoy et al. 2006).
A multitude of inflammatory mediators such as TNF-α, bradykinin, prostaglandins,
and cytokines are released following inflammation and reside in the affected peripheral
tissue. Furthermore, infiltrating immune cells such as mast cells, macrophages, and T
cells are also present in inflamed tissue. Many studies have investigated the role of
such cells and their secreted mediators in the development of sensitized nociceptors
following colitis. For example, supernatants obtained from colon biopsies of UC patients
have been shown to effectively reduce the rheobase of colonic DRG neurons, and
increase action potential discharge (Ibeakanma and Vanner, 2010). In the same study,
incubation with TNF-α mimicked these results, and TNF-α receptor deficient mice were
unresponsive to both supernatant and TNF-α incubations, indicating an important role
for TNFα in colitis-induced hyperalgesia. Furthermore, Barbara et al. (2007) obtained
12
supernatants from irritable bowel syndrome (IBS) patients and tested their effects on
Ca2+ mobilization in isolated DRG neurons in rats. They found that supernatants
obtained from IBS patients markedly enhanced cell excitability and action potential firing
rates in rat TRPV1+ DRG neurons exposed to capsaicin. When the antagonist for the
histamine receptor was administered prior to capsaicin activation, this effect was
reversed, implicating mast cell mediators as regulators of DRG sensitivity. Exogenous
material such as bacterial cell products can also induce peripheral sensitization, as
incubation of DRG neurons with lipopolysaccharide (LPS) significantly increases
neuronal excitability in acute and chronic incubation periods (Ochoa-Cortes et al. 2010).
Overall, peripheral sensitization arises from the actions of mediators in the
periphery leading to augmented function of various receptors and ion channels located
on the peripheral terminals of DRG nociceptors. The role of these factors have been
well examined, and it has been shown that peripheral sensitization occurs through both
direct and indirect (i.e. neurogenic inflammation) mechanisms. The results of peripheral
sensitization enhance nociceptive signaling and are likely to contribute to the
hyperalgesic (increased response to an already noxious stimulus) and allodynic
(response to an innocuous stimulus) responses of nociceptors.
1.2.3 Central Ascending Sensory Afferent Pathways in Pain Transmission
The major output from the dorsal horn to the brain is derived from projection
neurons located between laminae I and V (Basbaum and Jessell, 2000). Two classic
ascending pain pathways include the spinothalamic and spinoreticular tracts (Basbaum
et al. 2009). The spinothalamic tract relays pain signals from the dorsal horn to the
13
thalamus, whereas the spinoreticular tract relays information from the dorsal horn to the
brainstem. More recently, the dorsal column pathway has been described where
projection neurons synapse on the parabrachial region of the dorsolateral pons
(Palecek 2004). The output from this region connects with the amygdala, and it is the
amygdala that is considered to process information relative to the aversive properties of
pain (Basbaum et al. 2009). From the amygdala, structures such as the cingulate
cortex, somatosensory cortex, and the insular cortex are activated.
1.2.4 Sensitization of Ascending Pathways
The impact of inflammatory mediators on the development of peripheral
sensitization can in turn augment sensitivity at the level of the spinal cord, termed
central sensitization (Basbaum et al. 2009). In contrast to peripheral sensitization,
central sensitization has been less studied. Primarily, the release of neurotransmitters
from the central terminals of nociceptors into the synaptic cleft leads to post-synaptic
cell excitability (Murase and Randic 1984). When noxious stimuli are relayed, SP and
CGRP are released simultaneously with glutamate (Bueno and Fioramonti, 1999), and
SP has been shown to cause prolonged depolarization of dorsal horn neurons (Henry et
al. 1975; Murase and Randic 1984). This may be due to increased NK1R expression on
post-synaptic neurons following inflammation (Palecek et al. 2003).
Central sensitization can additionally arise from the disinhibition of descending
inhibitory pathways. Glycinergic or γ-aminobutyric acid (GABA)-ergic interneurons are
generally classified as inhibitory neurons. Studies have shown that the loss of inhibition
on dorsal horn projection neurons leads to behavioural hypersensitivity (Sivilotti and
14
Woolf, 1994; Malan et al. 2002). In a somatic inflammatory model using withdrawal
latency tests, Malan et al. (2002) showed that rats receiving GABA-receptor antagonists
became susceptible to tactile allodynia and thermal hyperalgesia. There has been
controversy surrounding whether disinhibition occurs by inhibitory neuron cell death, but
it is now recognized that cell death is not required for central sensitization to occur
(Moore et al. 2002; Polgar et al. 2005). Lastly, the psychological perception of
nociceptive signals is also a critical component to the generation of pain. The intensity
of pain experienced by patients has been shown to correlate to emotional
representation of illness, perceived stress levels, disease acceptance, and coping
(Kiebles et al. 2010).
Although central sensitization is an important component in the generation of
visceral hypersensitivity, this thesis is mainly concerned with changes occurring in
primary afferent DRG nociceptors. That being said, much of the data presented in this
thesis occurs at the level of the spinal cord where peripheral and central systems
converge, and the influence of peripheral sensitization on central sensitization was also
explored.
1.2.5 Nociceptors: Peripheral Versus Central Studies
As previously discussed, up-regulation of ion channel and receptor expression,
and increased inward cation current in peripheral nerve terminals increases action
potential discharge of nociceptors and enhances neuronal excitability following
inflammation. However, whether similar neurophysiological changes occur in the central
terminals of nociceptors following inflammation has not been investigated. In fact, the
15
role of nociceptor central nerve terminals in the development of visceral hypersensitivity
has only recently begun to be addressed (Harrington et al. 2012), and neuroanatomical
remodulation of these terminals may be an important mechanism underlying visceral
hypersensitivity following inflammation.
While the work described in this thesis was ongoing, Harrington et al. (2012)
used neuroanatomical tracing methods to determine whether colonic DRG nociceptor
terminals in the dorsal horn were sensitized following post-inflammatory colitis and
noxious colonic distension. They reported that nociceptor terminals of post-inflamed
animals were increased throughout the dorsal horn compared to healthy controls. In
addition, an increase in activated post-synaptic dorsal horn neurons was observed in
post-inflamed animals following colonic distension when compared to controls. These
findings support the contention that modulation of central terminals of DRG neurons
may also be important in sensitization of DRG neurons, but the mechanisms involved
remain poorly understood.
A fundamental mechanistic question concerns the nature of the inflammatory
response that leads to these changes. The findings from the above study did not
address the time dependent mechanisms, e.g. acute vs. chronic inflammation, that may
be involved in the onset of modulated nerve terminal endings in the dorsal horn. Thus,
studies of the time course of changes in nociceptor central nerve terminals in the
generation of visceral hypersensitivity are required. In addition, evidence for
mechanisms which could lead to enhanced post-synaptic cell activation in the dorsal
horn would provide additional functional relevance for the changes occurring in
peripheral DRG nociceptors.
16
1.3 Inflammation-Dependent Anti-Nociception of Pain Pathways
Inflammation-dependent regulation of pain is a poorly characterized dynamic
balance of pro-nociceptive and anti-nociceptive mechanisms. Pro-nociceptive mediators
promote the development and perpetuation of peripheral and central sensitization, two
essential components of visceral hypersensitivity. On the other hand, anti-nociceptive
mechanisms diminish the severity of peripheral and central sensitization, and promote
analgesia. The focus of this section is to characterize anti-nociceptive mediators that
regulate hypersensitivity and promote analgesia, and to discuss the role of ß-endorphin
in greater detail, including what is currently known of their role in IBD.
1.3.1 Inflammatory Mediators That Promote Analgesia
The viscera possess endogenous (mediators within the bowel wall) and
exogenous (luminal content) mechanisms that regulate analgesia (Agostini et al. 2009;
Mousa et al. 2010; Yamdeu et al. 2011) . Roles for endogenous molecules such as
NGF, enkephalins, nociceptin/orphanin FQ (NOFQ), receptors including intercellular
adhesion molecule 1 (ICAM1) and cannabinoid receptor 1 (CB1), and signaling
pathways such as the p38-MAPK pathway have all been identified to regulate antinociception (Agostini et al. 2009; Mousa et al. 2010; Yamdeu et al. 2011).
Agostini et al. (2009) looked at the effects of the endogenous opioid-like peptide,
NOFQ, on responses to colorectal distensions (CRD) using the TNBS model of colitis in
rats. They showed that peripherally administered NOFQ reduced the number of
abdominal contractions to CRD in TNBS-treated rats. This effect is likely mediated
17
through the opioid receptor-like receptor, ORL1, since administration of an ORL1
antagonist to TNBS-treated rats enhanced the pro-nociceptive effects of colitis.
NGF has been shown to increase anti-nociception via the p38-MAPK pathway
(Yamdeu et al. 2011). This was seen through an increase in µ-opioid receptor (MOR)
binding sites and MOR protein levels on DRG neurons, and an increase in the number
of MOR-immunoreactive DRG neurons. Additionally, enhanced ICAM1 expression via
sensory and sympathetic nerves is an important component to the recruitment of opioidcontaining immune cells and opioid-mediated analgesia (Mousa et al. 2010). Mousa et
al. 2010 reported that the degeneration of sensory and sympathetic nerves inhibited
enhanced ICAM1 expression after Freund’s complete adjuvant (FCA)-induced
inflammation and reduced the immigration of β-endorphin and enkephalin-containing
polymorphonuclear cells and mononuclear cells to inflamed regions. Furthermore, nerve
degeneration abolished opioid-mediated analgesia.
Our laboratory has recently shown that the concentration of the endogenous
opioid, β-endorphin, is increased in the colon following chronic DSS colitis and that the
DRG nociceptors are correspondingly less excitable (Valdez-Morales et al. 2012). This
was characterized in electrophysiological studies as a significant increase in rheobase
(the minimum current injection required to evoke discharge of a single action potential),
and a subsequent decrease in nociceptor firing activity at 2x the rheobase (Figure 3).
18
FIGURE 3. Incubation of naïve DRG neurons in supernatant from colons of chronic DSS-treated mice
decreases neuronal excitability.
Current-clamp patch clamp recordings of fast blue labeled colonic DRG neurons was used to assess
neuronal excitability. (A) Representative traces from naïve DRG neurons incubated overnight with
supernatant from either control or chronic DSS colons. (B) Summary data showing rheobase was
increased after overnight incubation with supernatant from chronic DSS supernatant (p = 0.0018). (C)
The number of action potentials at twice rheobase was significantly decreased after incubation with
supernatant from chronic DSS colons (p = 0.0230, Mann-Whitney test). Effects were reversed with
administration of naloxone (data not shown). From Valdez-Morales et al. (2012).
19
In addition, these anti-nociceptive effects of colitis were abolished with the
administration of the opioid receptor antagonist, naloxone, suggesting that opioids play
an important role in the regulation of nociception and the promotion of analgesia during
chronic inflammation in the colon. These findings may provide an explanation for
observed changes in nociceptive signaling in human IBD studies. It has been shown
that in acute flare-ups of UC patients exhibit visceral hyperalgesia. However, in patients
with chronic symptoms, i.e. for months to years, this hyperalgesia is no longer evident,
suggesting that anti-nociceptive factors may counteract the pro-nociceptive
mechanisms (Farthing and Lennard-Jones 1978; Bernstein et al. 1996).
1.3.2 β-Endorphin Regulation of Nociception
Enodorphins and dynorphins are families of opioid peptides that have a major
role in the promotion of analgesia. They bind to opioid receptors such as MOR, κ-OR
(KOR), and δ-OR (DOR) to elicit their anti-nociceptive effects. To date, the majority of
studies have investigated the role of a particular endorphin, β-endorphin, in the
regulation of nociception following somatic, and less frequently visceral, models of
inflammation (Mousa et al. 2001; Rittner et al. 2001; Verma-Gandhu et al. 2007).
Although β-endorphin has been shown to play an anti-nociceptive role in visceral
models of inflammation, the potential endogenous sources of this opioid have not been
reported. Possible sources of endogenous opioids include immune cells. For example,
in a model of FCA-induced inflammation of the hind paw, Mousa et al. (2001) reported
β-endorphin in CD4+ T cells. Using a similar inflammatory model, Rittner et al. (2001)
identified macrophages, monocytes, and granulocytes as opioid-producing leukocytes.
20
These cells were stained with 3E7 antibody to detect the N-terminus of opioid peptides.
In addition, this study separately quantified β-endorphin content in CD45+ cells
(hematopoietic cells) and showed that β-endorphin content increased in the inflamed
paw in a time dependent manner.
A subsequent study examined the effects of immunosuppression on antinociception using cyclosporin A-induced immunosuppression to cause a reduction in
circulating and infiltrating lymphocytes during an FCA-induced model of inflammation
(Hurmanussen et al. 2004). Nociceptive thresholds were decreased in
immunosuppressed rats, which could be reversed with administration of donor
lymphocytes. Verma-Gandhu et al. (2006) conducted a similar study in severe combined
immunodeficient (SCID) mice. SCID mice receiving reconstituted CD4+ T cells
displayed lower pain thresholds to CRD in the SCID mice were normalized.
Furthermore, this normalization was blocked by the opioid receptor antagonist,
naloxone, suggesting that T lymphocytes provided opioid-mediated anti-nociception. In
vitro experiments conducted in the same study showed the synthesis and release of βendorphin from stimulated T cells.
Studies of neuropathic pain have also characterized opioid-mediated antinociception. Labuz et al. (2010) used chronic constriction injury (CCI) of the sciatic
nerve and showed absence of an immune cell response in SCID mice to CCI. In
addition, this absence led to a decreased threshold of activation in somatic afferent
sensory nerves. In WT mice, CD3+/β-endorphin+ T cells comprised 11% of the
infiltrating immune cell population and when transferred into SCID mice, they fully
reversed the elevation in nociception. Moreover, corticotropin-releasing factor (CRF;
21
required for opioid release)-induced anti-nociception at the site of CCI was abolished
using antibodies targeted against β-endorphin and peripheral opioid receptors. An
earlier study using CCI of the sciatic nerve identified an infiltrating immune cell
population in which 30-40% contained opioids (Labuz et al. 2009). These cells were
CD45+/3E7+ immune cells and thus, suggest that opioids other than β-endorphin, such
as Met-enkephalin and dynorphin-A, are also contained in the infiltrating population.
Indeed, this is likely as it has been shown in other experiments that β-endorphin, Metenkephalin, and dynorphin-A are found in circulating lymphocytes and in lymph node
immune cells (Cabot et al. 2001). Furthermore, TNBS-induced colonic inflammation has
been reported to increase the proenkephalin 1 gene, the precursor for met- and leucineenkephalin (Kimball et al. 2007).
Few studies have used visceral models of inflammation to investigate opioidmediated anti-nociception in the GI tract. β-endorphin has been shown to be elevated
in blood samples from ulcerative colitis patients compared to controls (Kuroki et al.
2007). Similarly, rectal mucosal content of β-endorphin has been shown to be increased
in active UC patients, and decreased during remission (Yamamoto et al. 1996). In
patients with CD however, β-endorphin content in blood mononuclear leukocytes has
been shown to be decreased compared to control individuals (Wiedermann et al. 1994).
Experimentally, Verma-Gandhu et al. (2007) used dextran sulfate sodium (DSS) models
of acute and chronic colitis in mice to determine how the duration of colitis and
associating neuropeptide expression would affect pain. Key findings using CRD showed
that acute colitis mice had increased responsiveness to CRD with increases in SP and
IL-1β expression. On the other hand, chronic colitis mice showed no increase in
22
responsiveness to CRD, and less SP was present. Additionally, CD4+ T cells, and
increased expression of β-endorphin and the β-endorphin receptor, MOR, were only
identified in chronic colitis. The study concluded that acute colitis leads to hyperalgesia
whereas, chronic colitis-associated analgesia occurs as a result of increased βendorphin and MOR expression, and increased infiltrating lymphocytes.
Numerous studies have identified modulated opioid receptor expression in
models of colitis. Mustard oil-induced colitis causes a significant increase in DOR
mRNA expression within inflamed colon tissue (Kimball et al. 2007). Additionally, MOR
has been shown to have anti-inflammatory properties in the TNBS model of colitis,
where peripheral agonists of the receptor reduced inflammation (Philippe et al. 2003).
The effects were nearly abolished by naloxone and MOR-/- mice were highly susceptible
to colon inflammation (50% mortality rate after 3 days of TNBS administration). It should
also be noted that many models of somatic inflammation and nerve injury show similar
findings on modulated opioid receptor expression (Mousa et al. 2007, Verma-Gandhu et
al. 2007; Obara et al. 2009; Dubois et al. 2010; Lee et al. 2011). Thus, it is likely opioid
receptors play an important role in opioid-mediated anti-nociception, although direct
evidence for an immune source for these opioids in chronic inflammation in the colon
has been lacking.
1.4 Hypothesis
Our working hypothesis is that modulation of peripheral pain signaling in chronic
colitis results from a balance of pro-nociceptive and anti-nociceptive factors. The first
specific hypothesis was that pro-nociceptive factors would induce neuroanatomical
23
remodeling in the central nerve terminals of nociceptors that would increase nociceptive
signaling. The second specific hypothesis was that immune cells would be an important
source of endogenous opioids underlying the anti-nociceptive actions observed in
chronic inflammation.
1.4.1 Objectives Part 1: Pro-Nociception
To address our first hypothesis we measured if SP immunoreactivity, a marker for
peptidergic C fiber nociceptors, was increased in the dorsal horn of the spinal cord in a
DSS model of colitis that investigated different time courses. NK1R immunoreactivity in
post-synaptic dorsal horn cell bodies was also examined to identify if colitis could in turn
increase pro-nociceptive signaling on post-synaptic nerves in the spine.
1.4.2 Objectives Part 2: Anti-Nociception
To address our second hypothesis we used flow cytometry to investigate if
immune cell populations, specifically T cells and macrophages, expressed β-endorphin
following chronic DSS colitis in C57BL/6 mice.
24
Chapter 2
Methods
2.1 Animal Model
Studies were performed on adult (25-30 grams) male CD1 and adult (20-25
grams) male C57BL/6 mice obtained from Charles River Laboratories (Saint-Constant,
QC, Canada). All experimental protocols were established in accordance with the
regulations and guidelines of the Canadian Council on Animal Care and approved by
Queen’s University Animal Care Committee. Mice had ad libitum access to a standard
chow diet while maintained on a 12-hour light-dark cycle.
2.2 Introduction of Colitis
Experimental colitis was induced by administration of DSS (MW: 36,000-50,000,
MP Biomedicals, Solon, OH) in the drinking water. Acute colitis was induced by
administering 5% DSS for 5 days to CD1 mice, followed by a 2-day recovery period
during which normal drinking water was given. Chronic DSS in CD1 mice was achieved
through 3 cycles of DSS administration. In each cycle mice received 1.5% DSS for 5
days, followed by a 7-day recovery period of normal drinking water. Post-colitis
remodeling of afferent innervation was examined by administering an acute or chronic
DSS colitis protocol to CD1 mice, followed by an additional 5 week recovery period.
Chronic DSS in the C57BL/6 mouse strain was also produced through administration of
3 cycles of DSS feeding however, in each cycle 2% DSS was given for 5 days and a 5day recovery period of normal drinking water provided. The euthanization date for CD1
mice subjected to acute, chronic, post-acute, and post-chronic DSS colitis was on day
25
8, day 36, day 42, and day 71, respectively. The euthanization date for C57BL/6 mice
was on day 30. Control mice received normal drinking water for the duration of either
acute, chronic, or post-inflamed DSS treatments. Mice were monitored daily for signs of
behavioural distress, loose stool, rectal bleeding, and weight loss. Mice were
euthanized if weight dropped below 15% their initial weight during any DSS protocol.
One CD1 mouse following the post-chronic DSS protocol and one C57BL/6 mouse
following the chronic DSS protocol were euthanized due to excessive weight loss.
2.3 Immunohistochemistry
2.3.1 Tissue Cryosections
CD1 mice from control, acute, chronic, or post-inflamed DSS-treated groups
were anesthetized by intraperitoneal injection of ketamine/xylazine (0.166 mg/g) and
perfused transcardially using 4% paraformaldehyde (PFA; Sigma Aldrich Co., St. Louis,
MO). PFA perfusions were preceded by an injection of 0.1 mL heparin into the left
ventricle. The spinal cord segments T9-T13 and C2-C4 were dissected and fixed in 4%
PFA overnight at 4oC. Tissue was then transferred to 30% sucrose solution diluted in
PBS for 24 hours at 4oC and subsequently mounted in cryomatrix embedding medium
(Shandon Cryomatrix; Fisher Thermo Scientific, Mississauga, ON, Canada). Tissue was
frozen instantaneously in chilled 2-methylbutane (Mallinkrodt Baker Inc., Phillipsburg,
NJ) and stored at -80oC. Sections were cut using a cryostat (Cryotome® SME, Fisher
Thermo Scientific) and sections were 10 µm thick. All sections were collected on
Superfrost Plus microscope slides (Fisher Thermo Scientific).
26
Sections were washed three times in PBS-Tween (0.1%) before incubation in
10% normal donkey serum for 1 hour in a humidified chamber. Blocking serum was
removed, sections were washed with PBS-Tween, and the primary antisera: rat anti
mouse SP monoclonal antibody that detects the C terminus of the SP neuropeptide
(1:500; Fitzgerald Industries International Inc, Concord, MA) (Abbadie et al. 2001) and
rabbit anti mouse NK1R polyclonal antibody that detects the C terminus of the NK1R
protein (1:1000; Millipore, Billerica, MA) (Palecek et al. 2003) were added to the slides
overnight in a humidified chamber. This incubation was followed by 3 additional washes
in PBS-Tween and a 2-hour incubation in donkey anti-rabbit Dylight 549 (1:800;
Jackson ImmunoResearch, West Grove, PA) and/or donkey anti-rat Dylight 488 (1:800;
Jackson ImmunoResearch). Preliminary negative control experiments were conducted
to investigate the non-specific binding and fluorescence of secondary antisera; no
fluorescence was observed from either Dylight-conjugated 488 or 549 secondary
antiserum following the omission of the primary antiserum. Previous studies have
confirmed the specificity of NK1R primary antiserum as it has been reported that preabsorption of the NK1R antiserum with synthetic peptide corresponding with the C
terminus of NK1R results in the absence of immunofluorescent labeling (Piggins et al.
2001). In previous studies it has also been reported that pre-absorption of the SP
antiserum with SP or neurokinin A diminishes immunofluorescent labeling of the SP
antibody, but not when pre-absorbed with nerurokinin B (Wells et al. 1995; Talmage et
al. 1996). Slides were washed and covered with fluorescence mounting medium (Dako
North America Inc., Carpinteria, CA) or with fluorescence mounting medium containing
DAPI counterstain (Vector laboratories, Burlington, ON, Canada).
27
2.3.2 Quantification of Immunofluorescence
Each of the following steps were conducted in a blinded fashion where the
specific treatment to each tissue section and its associating micrograph(s) were
unknown to the investigator. SP immunoreactivity was visualized with an inverted Carl
Zeiss fluorescence microscope using a 20 x objective lens. Micrograph images were
acquired with Axio Vision camera software. Five fields of view obtained from three
sections of dorsal horn for every animal were analyzed using ImageJ software
(rsbweb.nih.gov/ij/), which was calibrated to yield regions of interest (ROI) in µm. SP
immunoreactivity was measured by adjusting pixel thresholds which converts
immunofluorescence into white pixels on a black background. To do this, a small area
containing the highest degree of background staining was selected in each micrograph
and was used as the threshold to distinguish between positively selected
immunoreactive pixels and negatively selected pixels. The selected region with the
highest background staining was converted to negative (black) pixels and the remaining
number of white immunoreactive pixels was quantified. ROI were 50 x 80 µm in size
and for every field of view, three ROIs were measured and averaged and ROI were
placed so that the 50 µm edge was along the start of laminae 1. In addition, ROI were
never overlapped during the time of analysis.
NK1R immunoreactivity was visualized with an Olympus BX51 microscope and
photographed using CoolSnap FX camera software. Images of spinal cord from control
and chronic DSS-treated mice were captured using the 40x objective lens. Three dorsal
horns were analyzed per animal. NK1 immunoreactivity of putative neurons was
28
quantified by counting the number of positively labelled cells that co-localized DAPIstaining of the cell nucleus in the dorsal horn.
2.4 Flow Cytometry
2.4.1 Lamina Propria Cell Isolation
This protocol was adopted and modified from Weigmann et al. (2007). C57BL/6
mice were split into two groups of control and chronic DSS-treated mice. Following the
chronic DSS protocol, mice were anesthetized in an isofluorane gas chamber and
subjected to cervical dislocation. The distal colon was isolated, surgically removed
excluding the cecum, and placed into chilled PBS. The colons from 4 control animals
were pooled together for each experiment date to ensure adequate numbers of immune
cells were present in the resulting isolated population. Similarly, 2 colons from DSStreated mice were pooled together each experiment date. Fewer colons were pooled in
chronic DSS conditions as the number of infiltrating immune cells increased at the site
of inflammation. A longitudinal incision was made along the colon wall and any feces
washed out. The colon was then cut into small, 1 mm thick pieces, and placed into a
PBS solution containing 5% FBS, 5 mM EDTA, and 1mM DTT for 1 hour at 37oC to
separate the lamina propria and muscle layer from epithelial, subepithelial, and villus
cells. Tissue was then washed with pre-warmed RPMI media containing 10% FBS and
placed into an RPMI enzymatic digestion solution containing 1 mg/mL collagenase D
(Roche, Laval, QC, Canada), 2 mg/mL Dispase II (Roche), and 1 mg DNAase 1 (Roche)
for 1 hour at 37oC to dissociate cells. Following the incubation period, cells were
washed with RPMI media and passed through a 40 µm cell strainer to isolate lamina
29
propria cells. Collected cells were centrifuged at 1000 rpm for 10 minutes at 4oC and
resuspended in a 40% percoll (GE Healthcare Bio-Sciences) solution. This solution was
carefully pipetted onto 80% percoll to establish a concentration gradient, and
centrifuged at 3800 rpm for 20 minutes at 4oC. Lamina propria immune cells suspended
between the two percoll concentrations were pipetted out, washed with RPMI, and
counted using a haemocytometer.
2.4.2 Magnetic Cell Sorting
All required materials were obtained from Miltenyi Biotec Inc., Auburn, CA. and
cells were kept on ice at all times during the experiment. Counted cells were centrifuged
at 1500 rpm for 10 minutes at 4oC and re-suspended in magnetic activated cell sorting
(MACS) buffer at a dilution of 1x107 cells: 90 µL MACS buffer. 10 µL of anti-CD4
microbeads: 1x107 cells was then added and cells incubated in microbeads for 15
minutes at 4oC. Cells were washed by centrifugation and re-suspended in 500 µL MACS
cell separation buffer. At this time two cell separation LS columns (one for control and
one for chronic DSS) were placed on the magnetic MACS multi stand and washed with
3 mL of chilled MACS buffer. LS columns contain a matrix of ferromagnetic spheres
covered in a non-toxic coating for cell separation. The re-suspended cells were then
passed through the column and allowed to filter by gravity. CD4+ cells adhered to
microbeads and remained inside the magnet of the LS column, whereas negatively
selected cells were filtered out. The columns were then removed from the multi stand
and positively selected CD4+ cells were obtained by flushing the column with 8 mL of
chilled MACS buffer, collected in a 15 mL conical tube. The above steps were then
30
repeated a second time using anti-CD11b microbeads on the negatively selected
population of cells.
2.4.3 Endogenous Opioid Labeling
Magnetically sorted CD4+ and CD11b+ cells from both control and chronic DSS
tissues were centrifuged at 1500 rpm for 10 minutes at 4oC, re-suspended into 500 µL
of 2% PFA, and plated in a 96-well round bottom plate at a density of 100,000-150,000
cells per well. Cells were then incubated in 2% PFA for 10 minutes at room temperature
followed by a wash by centrifugation at 2000 rpm for 3 minutes at 4oC. Cells were resuspended in 200 µL PBS and washed a second time. Cells were permeabilized
overnight in 50 µL of primary antiserum solution containing 5% BSA, 0.1% saponin, and
rabbit anti-β-endorphin antiserum (1:250, Millipore). Unstained and negative control
cells (treated only with secondary antiserum below) were kept in 50 µL PBS. Cells were
washed in PBS three times before a 1 hour incubation in 50 µL of goat anti-rabbit
phycoerythrin (PE)-conjugated secondary antiserum (1:100; Abcam, Cambridge, MA) at
room temperature. Unstained cells were kept in 50 µL PBS. Cells were washed three
times, resuspended in 150 µL PBS, and subjected to flow cytometry analysis.
2.4.4 Flow Cytometry
Single staining flow analysis was conducted on the Quanta SC flow cytometer
(Beckman Coulter, Mississauga, ON, Canada). Suspended cells from each well were
placed into the cytometer separately and analyzed for β-endorphin expression.
Unstained and negative control cells were used to set gating parameters to exclude cell
31
autofluorescence and background staining, respectively. Positively stained β-endorphin
cells were run through the cytometer and plotted on a fluorescence-side scatter plot to
analyze changes in β-endorphin expression in control and DSS-treated CD4+ and
CD11b+ cells.
2.5 Statistical Analysis
Statistical analyses were performed and graphs generated using GraphPad
Prism 5. Data are expressed as the mean ± SEM. Weight change data for DSS-treated
and control animals were compared using two-way ANOVA and Bonferroni post-hoc
tests. Immunoreactivity data were compared by Mann-Whitney or Kruskal-Wallis tests
f o l l o w e d b y D u n n ’s M u l t i p l e C o m p a r i s o n p o s t - t e s t s . F l o w c y t o m e t r y
immunofluorescence data was compared using the Mann-Whitney test. Statistical
significance was defined as P < 0.05.
32
Chapter 3
Results
3.1 Development of Colitis
Mice treated with DSS displayed distinctive symptoms of colitis including weight
loss, diarrhea, loose stool, and rectal bleeding. Weight charts for each mouse are
provided in Figures 4 through 7. Perfusion fixation of mice for immunohistochemical
analysis prevented quantitative biochemical assays of colitis severity, so the weight loss
observed in the mice treated with DSS was taken as evidence of the development of
colitis.
3.2 Controls
For all immunohistochemical studies, the optimal working dilution of each
antibody used was determined using manufacturer recommendations and serial
dilutions. The dilution that provided the best signal to noise ratio was employed for
immunohistochemical examinations. In addition, the omission of the primary or
secondary antibody resulted in the absence of labeling. For flow cytometry analysis,
negatively selected cell populations were labeled for CD4+ and CD11b+ cells. Results
showed that the negatively selected cells did not express CD4 and CD11b following
magnetic cell sorting. In addition, omission of the primary and secondary antibody
resulted in no fluorescence.
33
FIGURE 4. Weight change in Acute DSS-treated mice
Acute DSS mice were administered 5% DSS drinking water for 5 days followed by 2 days of normal
drinking water. Controls received normal drinking water. Weight change (grams) was measured daily for
each mouse and then averaged for each day. Acute DSS-treated mice showed significant weight loss on
days 5-7 of the Acute DSS protocol (* P < 0.05; *** P < 0.001). Statistical analysis was conducted using
2-way ANOVA and Bonferroni post-hoc tests.
34
FIGURE 5. Weight change in Post-acute DSS-treated mice
Post-acute DSS mice were administered 5% DSS drinking water for 5 days followed by 5 weeks of
normal drinking water. Controls received normal drinking water. Weight change (grams) was measured
daily for each mouse and then averaged for each day. Post-acute DSS-treated mice showed significant
weight loss on days 9-31 and on day 37 of the post-acute DSS protocol (* P < 0.05; where P<0.01 on
days 13-15,23-27,33-35 and P<0.001 on days 9,11,17,29,37). Statistical analysis was conducted using
2-way ANOVA and Bonferroni post-hoc tests.
35
FIGURE 6. Weight change in Chronic DSS-treated mice
Chronic DSS mice were administered 3 bouts of 5% DSS drinking water for 5 days followed by 7 days of
normal drinking water. Controls received normal drinking water. Weight change (grams) was measured
daily for each mouse and then averaged for each day. Chronic DSS-treated mice showed significant
weight loss on days 17-35 of the chronic DSS protocol (* P < 0.05; *** P < 0.001). Statistical analysis
was conducted using 2-way ANOVA and Bonferroni post-hoc tests.
36
FIGURE 7. Weight change in Post-chronic DSS-treated mice
Post-chronic DSS mice underwent chronic DSS, followed by an additional 5 weeks of recovery. Controls
received normal drinking water. Weight change (grams) was measured daily for each mouse and then
averaged for each day. Post-chronic DSS-treated mice showed significant weight loss on days 21-35 of
the post-chronic DSS protocol (* P < 0.05; *** P < 0.001). Statistical analysis was conducted using 2way ANOVA and Bonferroni post-hoc tests.
37
3.3. Results Part 1: Pro-Nociception
3.3.1 DSS-induced colitis increases DRG SP innervation in the dorsal horn of the
thoracic spinal cord
Nociceptive innervation of the spinal cord was assessed with SP
immunoreactivity, a neurotransmitter abundantly found in the C fiber subtypes of DRG
nociceptors that project to the superficial laminae (laminae I and II) of the dorsal horn
(Sugiura et al. 1986; Molliver et al. 1997; Bennett et al. 1998). CD1 mice underwent
DSS colitis that followed either an acute, post-acute, chronic, or a post-chronic model of
colitis. Age-matched controls received normal drinking water. Following acute DSS,
there was no significant change in SP immunoreactivity within the dorsal horn when
compared to control mice (Figure 8; n=5, P > 0.05). In contrast, a 4 fold increase in SP
immunoreactivity was observed within the dorsal horn (Figure 8; n=5 per group, P <
0.001) of mice 5 weeks following acute colitis, and following the induction of chronic
colitis. Post-acute and chronic DSS colitis protocols were both investigated, even
though they share similar time points of termination. However, this was done to address
whether hypothesized neuroanatomical remodeling can occur slowly following an acute
bout of inflammation, and/or following persistent inflammation. Similarly, the postchronic model of colitis was used to examine if neuroanatomical remodeling could
persist following recovery to chronic inflammation. A trend towards an increase in SP
immunoreactivity was observed in the post-chronic colitis group (Figure 8; n=5, P >
0.05). Age-matched control data were pooled together as mice euthanized on different
dates showed no statistical changes in SP immunoreactivity (data not shown).
38
A
LI
B
C
E
F
LII
LIII
LIV
D
FIGURE 8. Substance P (SP) immunoreactivity in the dorsal horn of the thoracic spinal cord segments T9T13 is increased following acute and chronic DSS colitis.
Mice received DSS drinking water to induce colitis or received normal drinking water as control.
Representative photomicrographs of SP immunoreactivity in the laminae (labeled LI - LIV in panel A) of
spinal cord segments T9-T13 were taken for control, acute, post-acute, chronic, and post-chronic DSStreated mice (A-E, respectively) and quantified using thresholded pixels per ROI (F). Following acute DSS
there was no significant change in SP immunoreactivity in the superficial laminae of the spinal cord (B;
P>0.05). In contrast, a significant increase of SP immunoreactivity was observed in both post-acute and
chronic DSS-treated mice (C and D). However following post-chronic DSS SP immunoreactivity was not
statistically different from control (F; P>0.05). Data summarized in F and tested for significance using the
Kruskal-Wallis and Dunns post-hoc tests. Significance was achieved when P < 0.05 (** P<0.01; ***
P<0.001). Scale bar = 30 ìm. Discrepancies in background staining were eliminated using pixel
thresholding during the quantification period. The dotted box in panel A indicates a representative ROI used
for analysis of immunoreactivity.
39
3.3.2 Colitis increases SP immunoreactivity in the central canal of the thoracic spinal
cord
Somatic models of inflammation such as formalin or capsaicin injection into the
hind paw of rodents have been shown to increase fos staining, a marker for cell
activation, in the dorsal horn (Jasmin et al. 1994; Palecek et al. 2003). Interestingly,
these studies showed fos staining was not limited to the superficial laminae but also
evident in lamina X, surrounding the central canal. The administration of exogenous
opioids can effectively reduce fos expression in lamina X (Jasmin et al. 1994),
suggesting the presence of presynaptic nociceptive nerve terminals in this region. In
addition, cholera toxin B subunit (CTB) tracing dye injected into the colon was observed
in DRG nerve terminals in this region following post-inflamed TNBS colitis (Harrington et
al. 2012). In the present study, we found marked increases in SP immunoreactivity
within lamina X of the dorsal horns obtained from CD1 mice with post-acute, chronic,
and post-chronic DSS colitis (Figure 9; n=5 per group, P < 0.001 post-acute and chronic
DSS; P < 0.01 post-chronic DSS). These changes in SP immunoreactivity were not
present in the acute DSS group of mice.
3.3.3 SP immunoreactivity is decreased in the cervical spinal cord segments C2-C4
following chronic DSS colitis
To determine if SP immunoreactivity was also elevated in nociceptors unexposed
to inflammation, segments of cervical spinal cord from chronic DSS treated mice were
dissected and labeled with the SP antiserum following colitis.
40
A
B
C
D
E
F
FIGURE 9. Substance P (SP) immunoreactivity in the central canal of the thoracic spinal cord segments T9T13 is increased following acute, chronic, and post-chronic DSS colitis.
Mice received DSS drinking water to induce colitis or received normal drinking water as control.
Representative photomicrographs of SP immunoreactivity near the spinal cord central canal in segments
T9-T13 were taken for control, acute, post-acute, chronic, and post-chronic DSS-treated mice (A-E,
respectively) and quantified using thresholded pixels per ROI (F). Following acute DSS there was no
significant change in SP immunoreactivity in the central canal (B; P>0.05). In contrast, a significant increase
of SP immunoreactivity was observed in post-acute, chronic, and post-chronic DSS-treated mice (C-E).
Data summarized in panel F and tested for significance using the Kruskal-Wallis and Dunns post-hoc tests.
Significance was achieved when P < 0.05 (** P<0.01; *** P<0.001). Scale bar = 30 ìm. Discrepancies in
background staining were eliminated using pixel thresholding during the quantification period. Dotted
circles indicate the location of the central canal.
41
Results showed that in contrast to thoracic spinal cord synapsing nociceptors,
nociceptor central terminals in the cervical spinal cord segments C2-C4 had decreased
SP immunoreactivity following chronic DSS colitis (Figure 10; n=5 per group, P < 0.05).
3.3.4 Effect of chronic DSS colitis on SP immunoreactivity in the colon
SP immunoreactivity was separately quantified in the mucosa and muscularis
externae of the distal colon following chronic DSS colitis to examine if peripheral nerve
terminals of DRG nociceptors also show increased SP immunoreactivity after a chronic
inflammatory stimulus. Results indicate that SP immunoreactivity in the distal colon is
unchanged within the muscularis externae following chronic DSS colitis (Figure 11; n=5
per group, P > 0.05). In addition, no statistical increase in SP immunoreactivity within
the mucosa of the distal colon following chronic DSS colitis was observed (Figure 11;
n=5 per group, P > 0.05).
3.3.5 NK1R is increased following chronic DSS on post-synaptic cells located in the
dorsal horn of the thoracic spinal cord.
The SP receptor, NK1R, is an important regulator of pain transmission. Previous
work has shown that increased NK1R-SP binding and internalization can exacerbate
and prolong pain (Murase and Randic, 1984; Honoré et al. 2002). We examined if
NK1R expression was increased on post-synaptic second order neurons in the spinal
cord following colitis. We found a 3 fold increase in NK1R immunoreactive cells in the
dorsal horn following chronic DSS colitis (Figure 12; n = 3, P < 0.05).
42
FIGURE 10. Substance P (SP) immunoreactivity in the dorsal horn of the cervical spinal cord segments
C2-C4 is decreased following chronic colitis.
Mice received DSS drinking water to induce chronic colitis or received normal drinking water as control.
Representative photomicrographs of SP immunoreactivity in the superficial laminae of cervical spinal
cord segments C2-C4 were taken for control and chronic DSS-treated mice (A and B, respectively) and
quantified using thresholded pixels per ROI (C). Following chronic DSS there a significant decrease in
SP immunoreactivity in the superficial laminae of the spinal cord (B; P = 0.0316). Data summarized in C
and tested for significance using the Mann-Whitney test (* P<0.05). Scale bar = 30 µm. Discrepancies in
background staining were eliminated using pixel thresholding during the quantification period.
43
FIGURE 11. Substance P (SP) immunoreactivity in the mucosa and muscularis externae of the distal
colon is unchanged following chronic colitis.
Mice received DSS drinking water to induce chronic colitis or received normal drinking water as control.
Representative photomicrographs of SP immunoreactivity in the distal colon were taken for control and
chronic DSS-treated mice (A and B, respectively) and quantified using thresholded pixels per ROI (C
and D). Following chronic DSS there was no statistically significant change in SP immunoreactivity
within the mucosa of the distal colon (C; P=0.0571). Likewise, no significant changes in SP
immunoreactivity were identified in the muscularis externae following chronic DSS colitis (D; P=0.6286).
Data was tested for significance using the Mann-Whitney test. Scale bar = 30 µm. Discrepancies in
background staining were eliminated using pixel thresholding during the quantification period.
44
Control
DSS
A
B
j
C
D
j
E
F
LI
LIV
LII
LIII
LIII
LII
LI
LIV
G
FIGURE 12. Neurokinin-1 Receptor (NK1R) immunoreactivity in the dorsal horn of the thoracic spinal cord
segments T9-T13 is increased following chronic colitis.
Slides were mounted using DAPI to label cell nuclei (A & B). Spinal cords were immunolabeled for NK1R from
control and chronic DSS mice (C & D respectively). Only cells with visible nuclei and staining clearly higher than
background levels were counted and representative photomicrographs show significantly elevated NK1R
immunoreactive cells throughout laminae I-IV (LI-LIV) in chronic DSS mice compared to control (E & F). White
arrowheads indicate examples of positively stained cells. Stars indicate cells shown in enlarged insets (widths 30
ìM). Data are summarized in panel G and were analyzed for significance using the Mann-Whitney test (P=0.0124; *
P < 0.05). Scale bar = 30 ìm.
45
Increased NK1R expression paired with increased SP immunoreactivity in the dorsal
horn suggests the potential for increased SP signaling in second order neurons and
increased post-synaptic excitability following chronic DSS colitis however, from this
immunohistochemical study it is difficult to determine if these increases result in
increased SP/NK1R complex internalization.
3.4 Results Part 2: Anti-Nociception
The following results were obtained from chronic DSS models of colitis using the
C57BL/6 strain of mice and examined anti-nociceptive mediators of colitis.
3.4.1 Chronic DSS colitis increases expression of the endogenous opioid β-endorphin in
lamina propria isolated CD4+ and CD11b+ immune cells
The colonic lamina propria is cellular, loose connective tissue that supports the
mucosal epithelium, and contains numerous immune cells which provide a secondary
line of defense to permeating antigens. Therefore, to investigate anti-nociceptive
mechanisms occurring following chronic DSS, specifically the potential immune cell
sources of the endogenous opioid, β-endorphin, CD4+ and CD11b+ immune cells from
the lamina propria were isolated and subjected to flow cytometry for single staining
analysis of expression for β-endorphin. In this study, both CD4+ and CD11b+ cell
populations showed an increase in the number of cells expressing β-endorphin following
chronic colitis. On average, CD4+ cells showed a 2.4 fold increase in β-endorphin
containing cells, whereas CD11b+ cells showed a 2.6 fold increase in β-endorphin
46
containing cells (Figure 13; n=5 CD4 control, n=7 CD4 chronic, n=4 CD11b control, n=5
CD11b chronic; P < 0.05 for CD4+ cells, P > 0.05 for CD11b+ cells).
47
FIGURE 13. β-endorphin is positively expressed in CD4+ and CD11b+ cells and is increased following
chronic DSS colitis
C57BL/6 mice were given DSS or normal drinking water. Lamina propria immune cells were dissociated
and magnetically cell sorted into CD4+ and CD11b+ cell populations. Cells were incubated in βendorphin primary antiserum and subjected to single staining flow cytometry analysis. β-endorphin was
positively expressed in control and DSS-treated CD4+ cells (A; left panel: control, right panel: chronic
DSS). Likewise, CD11b+ cells also expressed β-endorphin in control and DSS treated cells (B; left
panel: control, right panel: chronic DSS). Following chronic DSS colitis it was found that the number of
CD4+ cells expressing β-endorphin was significantly increased compared to control (C; P = 0.0303).
Following chronic DSS colitis, CD11b+ cells also showed a trend towards an increase in the number of
cells expressing β-endorphin however, these changes were not statistically significant (D; P = 0.0635).
Data were analyzed for significance using the Mann-Whitney test.
48
Chapter 4
Discussion
Pro-Nociception Factors: Alterations in Central Terminals of DRG Neurons
The major finding from the pro-nociception immunohistochemical studies we
conducted was that SP immunoreactivity in central DRG nerve terminals in the spinal
cord increased in a time-dependent manner following the induction of colitis. The
potential underlying mechanism(s) responsible for this observation may be related to
neuro-immune interactions (see below). Furthermore, an increase in NK1R
immunoreactivity was observed in post-synaptic projection neuronal cell bodies
following chronic colitis, which may have implications for the development of central
sensitization.
The immunoreactivity of SP in each micrograph analyzed was measured using
methods involving the adjustment of pixel thresholds. The major limitation of this
method is that a thresholded image results in each immunoreactive pixel displaying the
same intensity when in fact, a diverse range of pixelation intensities exist following
immunohistochemistry. The selection of each threshold was based on a subjective
interpretation of the image and discrepancies may have occurred. Another limitation
was that SP immunoreactivity was assumed to represent nerve terminals of DRG
neurons but this was not confirmed with double labeling studies using markers of axons
such protein gene product 9.5 (PGP).
4.1 Time-dependent increase of SP immunoreactivity in the thoracic spinal cord
segments T9-T13
49
SP was used as a marker for DRG nerve terminals in the dorsal horn since the
majority of colonic nociceptors are the C-fiber subtype that express CGRP and SP
(Robinson et al. 2004). Furthermore, electrophysiological recordings from colonic
afferent peripheral endings show these DRG neurons have high mechanosensory
thresholds (Brierley et al. 2004) and additionally are immunoreactive for the TRPV1 ion
channel (Brierley et al. 2005); two important markers of nociceptors. The SP
immunoreactivity following colitis suggests that time-dependent mechanisms are
important since no changes in SP immunoreactivity were observed in acute DSS.
However, five weeks following, a large increase in SP immunoreactivity was measured
in post-acute DSS mice. Furthermore, increased SP immunoreactivity was also
observed in chronic DSS mice and five weeks later in post-chronic DSS mice.
The gut contains a vast supply of innate and adaptive immune factors, some of
which are completely unique to the gut (Macdonald and Monteleone, 2005). Primarily,
the gut epithelial barrier provides innate, structural protection against luminal antigens
and works to prevent antigens from crossing over into subsequent tissues. However, the
complete exclusion of antigens by the epithelial barrier does not occur. Follicleassociating epithelium (FAE) overlying aggregates of lymphoid tissue called Peyer’s
Patches (PP) contain specialized epithelial cells that function to transport luminal
antigens into PP. Here antigens are presented to numerous immunocytes such as B
cells, T cells, macrophages, and dendritic cells. Furthermore, dendritic cells also send
processes between gut epithelium to sample bacteria. This region is thus considered to
be part of the epithelial barrier that is specialized for antigen uptake. In regions of the
epithelium where FAE and PP are absent, aberrant permeability to antigens may induce
50
a subsequent aberrant immune response. When inflammation occurs, such as in
chronic colitis, antigens leak through the epithelial barrier and trigger T cell activation.
Immunosuppressive cytokines and mediators such as T regulatory cells are overrun by
aberrant functioning of the T cell immune response where increased release of proinflammatory cytokines TNF-α and IFN-γ cause tissue damage and further increase
epithelial permeability. It is this eventual tissue damage caused by inflammation that
may reflect the time dependent increases seen in SP immunoreactivity we observed.
In addition, our time dependent results support the hypothesis that
neuroanatomical remodeling occurs following colitis as ongoing inflammation associates
with increased peripheral tissue damage, and tissue damage has been shown to induce
axonal outgrowth in studies of neuropathy. Colonic inflammation has been shown to
present with enteric neuropathy (Tornblom et al. 2002; reviewed in Lomax et al. 2006)
but whether neuropathy occurs specifically in colonic nociceptors has not been
investigated.
NGF is a neurotrophin that is responsible for the growth, maintenance and
survival of neurons, especially during development. Hu et al. (2011) used in vitro coculture models of DRG neurons and Schwann cells containing NGF to investigate
axonal outgrowth. Substantial neurite outgrowth of DRG neurons was observed in both
the co-culture model as well as in a model that placed DRG neurons alone into
Schwann cell-conditioned media. Furthermore, when the NGF receptor inhibitor K-252a
was applied, DRG neurite outgrowth was significantly decreased in a time-dependent
manner. Additionally, Averill et al. (1995) showed that the NGF high affinity receptor,
trkA, is specifically expressed on the C fiber subtype of colonic DRG nociceptors that
51
also utilize CGRP and SP. When bound, trkA activates a signaling cascade, ultimately
resulting in regulation of various transcriptional processes within the cell nucleus, one of
which is responsible for neurite growth. At this stage, time-dependent regulation of DRG
nociceptor sensitivity may be occurring if the processing of activated transcriptional
activity into resultant central terminal remodulation is slow.
SP innervation of the spinal cord is increased following chronic and post-inflamed
models of colitis but the mechanism or mechanisms underlying this change have yet to
be identified. There are 3 potential mechanisms for increased innervation of the spinal
cord; increased neurotransmitter synthesis in nociceptor terminals, increased axonal
outgrowth and the development of new central terminals, and/or the recruitment of silent
nociceptors (Figure 14). While we cannot rule out any of these mechanisms at present,
there is some data to suggest that neuroanatomical remodeling (axonal outgrowth)
accounts for increased SP immunoreactivity in the spinal cord that was observed.
4.1.1 Evidence for neuroanatomical remodeling
A recent study by Harrington et al. (2012) supports the hypothesis that
neuroanatomical remodeling of nociceptors occurs following colitis. In this study, the
TNBS model of post-inflammatory colitis was used. The colonic afferent central
terminals of DRG neurons were retrogradely labeled using CTB dye injected into the
colon. They found that there was an increase in the mean area of dorsal horn with the
presence of CTB staining following post-inflammatory TNBS colitis. In addition, CTB
staining was present in lamina I, V, and near the central canal, regions of dorsal horn
where nociceptors are known to synapse.
52
FIGURE 14. Potential mechanisms for increased SP innervation of the spinal cord
The increases observed in SP immunoreactivity following colitis may be attributable to three different
mechanisms. Primarily, increased SP immunoreactivity may be resultant of increased SP synthesis within
already present nociceptors (upper panel). In addition, increased SP innervation may arise from the
remodeling of nociceptor central terminals, where an increase in axonal outgrowth occurs (middle panel).
Furthermore, the recruitment of previously silent nociceptors into active nociceptors might occur following
colitis and increase SP innervation of the spinal cord (bottom panel). It is also possible that previously
innocuous sensory afferents undergo a phenotypic switch following inflammation, and begin to synthesize
detectable levels of SP which may ultimately contribute to the increases in SP innervation that was
observed (bottom panel).
53
These results are consistent with the locations of SP immunoreactivity in the dorsal horn
observed in the work of this thesis. Indeed, Harrington et al. (2012) also showed that
CTB staining was co-localized with CGRP and SP. Together, these data suggest that
CTB-labeled colonic central afferent terminals observed belong to colonic nociceptors.
Together, this data suggests that central terminals of colonic nociceptors can undergo
neuroanatomical remodeling following post-inflammatory TNBS colitis and possibly
other inflammatory insults.
In addition, recent preliminary in vitro studies also support this hypothesis
(personal communication, Dr. C. Altier). Here, following an acute DSS model of colitis
where C57BL/6 mice received 2% DSS drinking water for 7 days, DRG T10-T12 and
L6-S1 were removed, cultured overnight, and stained for β-tubulin, a marker for cellular
membrane. Results showed drastic changes between β-tubulin staining in control
versus DSS-treated cells. DRG neurons from DSS-treated mice showed much more
axonal sprouting than control DRG neurons following 24 hours of cell culture. Similarly,
when a neuronal cell line was incubated with TNF-α and stained for β-tubulin, TNF-α
induced observable neurite outgrowth in the cells.
4.2. SP immunoreactivity in colonic tissue
DRG nociceptors are pseudo-unipolar and thus it is possible that excess SP
synthesized in the cell bodies of these neurons is trafficked equally to both central and
peripheral terminals. Therefore, SP immunoreactivity was measured in the distal colon,
where peripheral terminals of DRG nociceptors project.
54
To date, controversial evidence exists for SP immunoreactivity in the colon during
IBD as both increases and decreases in SP immunoreactivity have been observed
(Mazumbar and Das 1992; Karanen et al. 1995; Wantanabe et al. 1997; Miampamba
and Sharkey 1998; Auli et al. 2008). Furthermore, the contribution of DRG nociceptors
to SP immunoreactivity in the colon following colitis are unexplored. Because SP is
expressed in numerous cell types including immune cells, epithelial cells, and enteric
neurons, it is difficult to specify the role of peripheral DRG nociceptor terminals. In
addition, the locations of peripheral DRG nociceptor terminals are difficult to study
anatomically. Following chronic DSS colitis in CD1 mice we found a trend towards an
increase in SP immunoreactivity within the mucosa of the colon, although statistical
significance was not achieved. However, the increase observed in the mucosa was
associated with the presence of large numbers of infiltrating SP-containing immune cells
and it was not possible to specifically examine axons. On the other hand, no change in
SP immunoreactivity was observed in the muscularis externae.
4.3 SP immunoreactivity in the cervical spinal cord
SP immunoreactivity was measured in the cervical spinal cord segments C2-C4
as a negative control to determine if inflammation of the colon could in turn also
increase the expression of SP in DRG nociceptors located proximally, in the absence of
tissue damage. Surprisingly, a significant decrease in SP immunoreactivity was
observed following chronic DSS colitis. One possible mechanism to explain this could
be that circulating cytokines resultant from colonic inflammation increase SP release
from proximally located DRG neurons by activating them at peripheral terminals, which
55
would decrease the amount of SP in central terminals when compared to controls.
However, this data also implies that the increased SP immunoreactivity observed in
thoracic spinal cord segments is a localized phenomenon, and during colitis only DRG
nociceptors directly exposed to inflammation exhibit this increase.
4.4 NK1R immunoreactivity is increased in the thoracic spinal cord following chronic
colitis
. NK1R is one of three neurokinin G protein-coupled receptors and it binds SP
with the highest affinity (Helke et al. 1990). Upon activation by ligand binding NK1R will
in turn activate cell signaling cascades that will ultimately lead to the phosphorylation of
various ion channels that will depolarize the cell and generate action potential discharge
(King et al. 1997). Our findings that up-regulation of NK1R occurs on putative postsynaptic dorsal horn projection neurons in the thoracic dorsal horn following chronic
DSS colitis in addition to the changes observed in SP immunoreactivity may have
important implications in the onset of central sensitization, and additionally play an
important role in the development of visceral hypersensitivity. A limitation of these NK1R
immunohistochemical studies was that we presumed that staining involved neurons but
did not exclude the possibility of staining glial cells.
Previous work in somatic inflammatory models have shown that NK1R mRNA
levels in the dorsal horn of mice are elevated following formalin or FCA-induced
peripheral inflammation of the hind paw (Palecek et al. 2003). The increases in NK1R
mRNA was highest at later time points (24 hours and 4 days) following inflammation by
formalin and FCA, respectively. This suggests that NK1R may be an important mediator
56
in the development of central sensitization in post-synaptic dorsal horn projection
neurons following inflammatory states. In our study we found an increase in NK1R
protein immunoreactivity in the dorsal horn of the thoracic spinal cord compared to
NK1R in healthy controls. Whether or not increased NK1R immunoreactivity following
inflammation correlates with increased SP uptake and internalization involves further
experiments.
Increased NK1R immunoreactivity may be particularly relevant for the post
synaptic dorsal column (PSDC) central ascending pathway. Palecek et al. (2003)
retrogradely labeled PSDC neurons with fluorescent dye injected into the gracile
nucleus, a previously described termination point of PSDC-projecting axons. Six hours
following administration, NK1R immunoreactivity was examined on PSDC nerve
endings in the dorsal horn. They reported that PSDC neurons were located mainly in
laminae III-IV and around the central canal region in T11-T13. Approximately 3% of
PSDC neurons in T11-T13 positively expressed NK1R following inflammation whereas
control animals showed no NK1R labeling on PSDC neurons. Thus, there may be a
functional role for central sensitization of the PSDC central ascending pathway following
colitis. However, both Palecek et al. (2003) and our findings showed that NK1R
expression was not limited to laminae III-IV and the central canal. Instead, NK1R
immunoreactivity was seen in most laminar regions which suggests that increased
NK1R immunoreactivity following chronic DSS or acute zymosan inflammation may not
be dependent on PSDC neurons alone.
When SP binds NK1R, activation of the receptor and downstream signaling
occurs. A SP/NK1R complex is also formed and internalized into the cell, resulting in
57
desensitization and inactivation of receptor signaling (Bowden et al. 1994; Adelson et al.
2009). The results of this study do not differentiate between membrane bound and
internalized NK1R however, this could be examined in the future.
4.5 Future studies for pro-nociceptive mechanisms
Our pro-nociceptive study provided new insight into the regulation of visceral
hypersensitivity at the interface of peripheral and central neural systems convergence in
the spinal cord. We observed an increase in SP innervation of the thoracic spinal cord
and following experiments are required to investigate the plausible mechanisms
underlying these observations. Immunohistochemical analysis of SP immunoreactivity in
the dorsal horn double-labeled with GAP43, a marker of new neuronal terminals, would
distinguish if increased SP immunoreactivity is resultant of the development of new
nociceptor terminals or from increased synthesis from existent terminals. Preliminary
work was done in this study to investigate this outcome however the specificity of the
GAP43 antibody used (1:1500; Millipore) was non-specific and fluorescently labeled
post-synaptic neuronal cell bodies (data not shown). In addition, an axonal neural
filament marker such as PGP could be used to identify axons in the spinal cord
(Lundberg et al. 1988) and to confirm that SP immunoreactivity is contained within
axons.
The use of confocal microscopy would be very helpful to better understand if
increased NK1R immunoreactivity in the dorsal horn is the result of increased
expression on the cell membrane or from increased internalization. In addition, the use
of other models to examine functional changes to post-synaptic cell excitability can be
58
paired with NK1R confocal microscopy examination. Specifically, colitis models using
noxious CRD to activate the nociceptor pathway, paired with immunohistochemical
analysis of pERK1/2 immunoreactivity in post-synaptic dorsal horn projection neurons
would identify whether colitis leads to increased post-synaptic cell activity compared to
control following a noxious stimulus.
Anti-Nociception Factors: Opioid-Containing Immune Cells in Colitis
In addition to examining pro-nociceptive mechanisms involved in the
development of peripheral and central sensitization, we aimed to examine the source of
the anti-nociceptive endogenous opioid, β-endorphin, during chronic DSS colitis.
Although many somatic models of inflammation have shown that β-endorphin is an
important regulator of analgesia following inflammation, there has been very little study
in visceral organs and in chronic models of inflammation. This is of particular interest in
the intestine given the unique immune system in the GI tract and the potential role of
immune cells as an important source of β-endorphin. As previously discussed, our
laboratory has shown the importance of β-endorphin levels as an anti-nociceptive factor
in chronic DSS colitis and in human ulcerative colitis (Valdez-Morales et al. 2012).
Although studies suggest that β-endorphin is important, the exact cellular sources of this
endogenous opioid are not clear. Thus the objective of this thesis was to examine if T
cells and macrophages contribute to analgesic properties of DRG neurons in chronic
DSS colitis through modulation with β-endorphin.
4.6 Sources of β-endorphin in chronic DSS colitis
59
This thesis found that both T cells and macrophages express β-endorphin and
that the number of T cells and macrophages positively expressing β-endorphin
significantly increases following chronic colitis. These results suggest that both T cells
and macrophages could be important sources of β-endorphin during chronic
inflammation, and that they may be important for the anti-nociceptive regulation of DRG
nociceptor hypersensitivity following the induction of colitis.
The results obtained in this thesis are supported by evidence from studies of the
somatic nervous system examining the roles of β-endorphin-containing immune cells in
the regulation of nociception. T cells and macrophages have been identified as opioidcontaining immune cells following inflammation in somatic models (Mousa et al. 2001;
Rittner et al. 2001). More recently, Verma-Gandhu et al. (2006) used CRD to compare
the visceromotor responses of SCID and control mice. Here, SCID mice exhibited
significantly lower pain thresholds however, when reconstituted with CD4+ T cells, the
visceral pain response was normalized. In addition, the effects of T cell reconstitution in
SCID mice were blocked using naloxone, the opioid receptor antagonist. Further studies
have determined that the duration of colitis is related to the abundance of antinociceptive mediators present at a given time (Verma-Gandhu et al. 2007). Specifically,
this work showed that in acute DSS colitis, mice had increased responsiveness to CRD
and increases in content of pro-nociceptive mediators such as SP and interleukin-1
(IL-1). In contrast, following chronic DSS colitis, mice were no longer hyper-responsive
to CRD. Also following chronic DSS colitis, β-endorphin content and MOR content were
up-regulated. Thus, evidence from studies of somatic nerves suggests that indeed, βendorphin may play an important role in regulating nociception following colitis, and that
60
the source of β-endorphin is provided by CD4+ or CD11b+ immune cells, such as T
cells and macrophages.
CD11b was used as a marker for macrophages in this study however, CD11b is
also a marker on other immune cells such as granulocytes, natural killer cells, and
dendritic cells (Cerovic et al. 2012; Johnson et al. 2012). Thus, the specificity of the
CD11b results may not be restricted to macrophages only. Further investigation using a
second marker of macrophages such as F4/80 (Neshat et al. 2009; Schiechl et al. 2011)
in the magnetic cell sorting process may be beneficial. That being said, somatic
inflammatory models have shown that macrophages are an endogenous source of
opioids and thus, likely play a regulatory role in peripheral nociception (Rittner et al.
2001). In addition, we also investigated CD11c+ dendritic cells (Cerovic et al. 2012), as
a potential source of β-endorphin and our results show that β-endorphin content in
CD11c+ cells does not change following chronic colitis (data not shown). Thus, if nonmacrophage CD11b+ immune cells play an important role in the up-regulation of βendorphin following chronic DSS colitis, the role of dendritic cells can likely be ruled out.
4.7 Future studies of anti-nociceptive mechanisms
A series of future experiments to better our understanding of anti-nociceptive
sources in colitis involve investigations relating to opioid release from cells, opioid
receptor expression, and the anti-inflammatory properties of β-endorphin. Primarily, the
use of cell culture and ELISA can be used to explore the release properties of βendorphin. Using magnetically cell separated CD4+ and CD11b+ cells from control
mice, the release of β-endorphin can be measured by ELISA following incubation in
61
supernatant obtained from chronic DSS mice. If changes are seen between supernatant
cultured cells and controls, then further investigation into what specific mediators trigger
β-endorphin release from immune cells can be investigated.
Secondly, investigating whether up-regulation of the opioid receptors occurs
following chronic DSS colitis would determine if increased β-endorphin content and
release in the colon during colitis is paired with increased receptor expression, a likely
indicator of increased receptor-ligand binding, on peripheral DRG nerve terminals.
Immunohistochemistry targeting MOR and markers for nociceptor terminals such as
CGRP and TRPV1 could be used. Reverse transcriptase PCR studies investigating
mRNA levels of MOR in colon-innervating DRG neurons is also plausible.
Lastly, roles for anti-inflammatory effects of β-endorphin have also been
described through investigations looking at MOR in TNBS colitis. Specifically, work by
Philippe et al. (2003) showed that following TNBS colitis, agonists of MOR, DALDA and
DAMGO, significantly reduced inflammation evidenced by improved macroscopic
lesions, decreased neutrophil infiltrate, absence of ulceration, and decreased MPO
scores. These effects were almost completely abolished by concomitant administration
of naloxone, suggesting a dual role for β-endorphin in the regulation of both nociception
and inflammation. Thus, examination of whether β-endorphin binding to MOR can elicit
anti-inflammatory effects is a promising path of investigation; diminishing inflammation
and the associating presence of pro-inflammatory cytokines may ultimately promote
anti-nociception and provide further insight into how β-endorphin regulates nociception
in the colon following colitis.
62
4.8 Concluding Remarks
In conclusion, we have addressed the multiple pro-nociceptive and antinociceptive mediators involved in the development and regulation of peripheral and
central sensitization following GI inflammation. Although clear evidence exists to show
that pro-nociception is enhanced following inflammation, we also report an important
role for anti-nociceptive factors. It is likely that a balance exists between pro- and antinociception and depending on the individual, severity of disease, and environmental
factors, this balance is unique to each individual (Figure 15). As a result, better
understanding of the development and regulation of peripheral and central sensitization
is needed.
Although the nociceptive pathway is complex
potential therapeutic intervention.
63
it offers a major site for
FIGURE 15. Nociception is determined by a balance of pro- and anti-nociceptive mediators
Nociception in the colon is a dynamic balance of pro-nociceptive and anti-nociceptive factors that are
unique to each individual. In turn, each individual will have their own, unique version of the scale depicted
here, where pro-nociceptive and anti-nociceptive factors regulate visceral hypersensitivity.
64
Reference List
Abbadie C, Pasternak GW, and Aicher SA (2001). Presynaptic localization of the
carboxy-terminus epitopes of the mu opioid receptor splice variants MOR-1C and
MOR-1D in the superficial laminae of the rat spinal cord. Neuroscience 106,
833-842.
Adam B, Liebregts T, Gschossmann JM, Krippner C, Scholl F, Ruwe M, and
Holtmann G (2006). Severity of mucosal inflammation as a predictor for
alterations of visceral sensory function in a rat model. Pain 123, 179-186.
Adelson D, Lao L, Zhang G, Kim W, and Marvizon JC (2009). Substance P
release and neurokinin 1 receptor activation in the rat spinal cord increase with
the firing frequency of C-fibers. Neuroscience 161, 538-553.
Agostini S, Eutamene H, Broccardo M, Improta G, Petrella C, Theodorou V, and
Bueno L (2009). Peripheral anti-nociceptive effect of nociceptin/orphanin FQ in
inflammation and stress-induced colonic hyperalgesia in rats. Pain 141, 292-299.
Aigner L, Arber S, Kapfhammer JP, Laux T, Schneider C, Botteri F, Brenner HR,
and Caroni P (1995). Overexpression of the neural growth-associated protein
GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice.
Cell 83, 269-278.
Akbar A, Yiangou Y, Facer P, Brydon WG, Walters JR, Anand P, and Ghosh S
(2010). Expression of the TRPV1 receptor differs in quiescent inflammatory
bowel disease with or without abdominal pain. Gut 59, 767-774.
Almeida TF, Roizenblatt S, and Tufik S (2004). Afferent pain pathways: a
neuroanatomical review. Brain Res 1000, 40-56.
Ament ME (1975). Inflammatory disease of the colon: ulcerative colitis and
Crohn's colitis. J Pediatr 86, 322-334.
Auli M, Nasser Y, Ho W, Burgueno JF, Keenan CM, Romero C, Sharkey KA, and
Fernandez E (2008). Neuromuscular changes in a rat model of colitis. Auton
Neurosci 141, 10-21.
65
Averill S, McMahon SB, Clary DO, Reichardt LF, and Priestley JV (1995).
Immunocytochemical localization of trkA receptors in chemically identified
subgroups of adult rat sensory neurons. Eur J Neurosci 7, 1484-1494.
Bang H, Kim Y, and Kim D (2000). TREK-2, a new member of the
mechanosensitive tandem-pore K+ channel family. J Biol Chem 275, 1741217419.
Barbara G, Wang B, Stanghellini V, de GR, Cremon C, Di NG, Trevisani M,
Campi B, Geppetti P, Tonini M, Bunnett NW, Grundy D, and Corinaldesi R
(2007). Mast cell-dependent excitation of visceral-nociceptive sensory neurons in
irritable bowel syndrome. Gastroenterology 132, 26-37.
Basbaum A & Jessell T (2000). The perception of pain. In Principles of Neural
Science, eds. Kandel ER, Schwartz JH, & Jessell TM, McGraw-Hill, Health
Professions Division, New York.
Basbaum AI, Bautista DM, Scherrer G, and Julius D (2009). Cellular and
molecular mechanisms of pain. Cell 139, 267-284.
Bennett DL, Averill S, Clary DO, Priestley JV, and McMahon SB (1996).
Postnatal changes in the expression of the trkA high-affinity NGF receptor in
primary sensory neurons. Eur J Neurosci 8, 2204-2208.
Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q,
McMahon SB, and Priestley JV (1998). A distinct subgroup of small DRG cells
express GDNF receptor components and GDNF is protective for these neurons
after nerve injury. J Neurosci 18, 3059-3072.
Benowitz LI and Routtenberg A (1997). GAP-43: an intrinsic determinant of
neuronal development and plasticity. Trends Neurosci 20, 84-91.
Bernstein CN, Niazi N, Robert M, Mertz H, Kodner A, Munakata J, Naliboff B,
and Mayer EA (1996). Rectal afferent function in patients with inflammatory and
functional intestinal disorders. Pain 66, 151-161.
Bessou P and Perl ER (1969). Response of cutaneous sensory units with
unmyelinated fibers to noxious stimuli. J Neurophysiol 32, 1025-1043.
66
Beyak MJ, Bulmer DCE, Jiang W, Keating C, Rong W, & Grundy D (2006).
Extrinsic Sensory Afferent Nerves Innervating the Gastrointestinal Tract. In
Physiology of the Gastrointestinal Tract, ed. Johnson LR, pp. 685-725. Academic
Press.
Beyak MJ, Ramji N, Krol KM, Kawaja MD, and Vanner SJ (2004). Two TTXresistant Na+ currents in mouse colonic dorsal root ganglia neurons and their
role in colitis-induced hyperexcitability. Am J Physiol Gastrointest Liver Physiol
287, G845-G855.
Bowden JJ, Garland AM, Baluk P, Lefevre P, Grady EF, Vigna SR, Bunnett NW,
and McDonald DM (1994). Direct observation of substance P-induced
internalization of neurokinin 1 (NK1) receptors at sites of inflammation. Proc Natl
Acad Sci U S A 91, 8964-8968.
Brierley SM, Jones RC, III, Gebhart GF, and Blackshaw LA (2004). Splanchnic
and pelvic mechanosensory afferents signal different qualities of colonic stimuli in
mice. Gastroenterology 127, 166-178.
Brierley SM, Carter R, Jones W, III, Xu L, Robinson DR, Hicks GA, Gebhart GF,
and Blackshaw LA (2005). Differential chemosensory function and receptor
expression of splanchnic and pelvic colonic afferents in mice. J Physiol 567, 267281.
Bueno L and Fioramonti J (1999). Effects of inflammatory mediators on gut
sensitivity. Can J Gastroenterol 13 Suppl A, 42A-46A.
Cabot PJ, Carter L, Schafer M, and Stein C (2001). Methionine-enkephalin-and
Dynorphin A-release from immune cells and control of inflammatory pain. Pain
93, 207-212.
Carroccio A, Mansueto P, Iacono G, Soresi M, D'Alcamo A, Cavataio F, Brusca I,
Florena AM, Ambrosiano G, Seidita A, Pirrone G, and Rini GB (2012). NonCeliac Wheat Sensitivity Diagnosed by Double-Blind Placebo-Controlled
Challenge: Exploring a New Clinical Entity. Am J Gastroenterol.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, and Julius D
(1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway.
Nature 389, 816-824.
67
Caterina MJ, Rosen TA, Tominaga M, Brake AJ, and Julius D (1999). A
capsaicin-receptor homologue with a high threshold for noxious heat. Nature
398, 436-441.
Cattaruzza F, Lyo V, Jones E, Pham D, Hawkins J, Kirkwood K, Valdez-Morales
E, Ibeakanma C, Vanner SJ, Bogyo M, and Bunnett NW (2011). Cathepsin S is
activated during colitis and causes visceral hyperalgesia by a PAR2-dependent
mechanism in mice. Gastroenterology 141, 1864-1874.
Cerovic V, Houston SA, Scott CL, Aumeunier A, Yrlid U, Mowat AM, and Milling
SW (2012). Intestinal CD103(-) dendritic cells migrate in lymph and prime
effector T cells. Mucosal Immunol.
Chapman CD, Ammons WS, and Foreman RD (1985). Raphe magnus inhibition
of feline T1-T4 spinoreticular tract cell responses to visceral and somatic inputs.
J Neurophysiol 53, 773-785.
Clarke K, Sagunarthy R, and Kansal S (2008). RDW as an additional marker in
inflammatory bowel disease/undifferentiated colitis. Dig Dis Sci 53, 2521-2523.
Crohn's and Colitis Foundation of Canada. Annual Report 2007-2008. 2008.
Delafoy L, Raymond F, Doherty AM, Eschalier A, and Diop L (2003). Role of
nerve growth factor in the trinitrobenzene sulfonic acid-induced colonic
hypersensitivity. Pain 105, 489-497.
Delafoy L, Gelot A, Ardid D, Eschalier A, Bertrand C, Doherty AM, and Diop L
(2006). Interactive involvement of brain derived neurotrophic factor, nerve growth
factor, and calcitonin gene related peptide in colonic hypersensitivity in the rat.
Gut 55, 940-945.
Deval E, Gasull X, Noel J, Salinas M, Baron A, Diochot S, and Lingueglia E
(2010). Acid-sensing ion channels (ASICs): pharmacology and implication in
pain. Pharmacol Ther 128, 549-558.
Dubois D and Gendron L (2010). Delta opioid receptor-mediated analgesia is not
altered in preprotachykinin A knockout mice. Eur J Neurosci 32, 1921-1929.
68
Farthing MJ and Lennard-jones JE (1978). Sensibility of the rectum to distension
and the anorectal distension reflex in ulcerative colitis. Gut 19, 64-69.
Gaudio E, Taddei G, Vetuschi A, Sferra R, Frieri G, Ricciardi G, and Caprilli R
(1999). Dextran sulfate sodium (DSS) colitis in rats: clinical, structural, and
ultrastructural aspects. Dig Dis Sci 44, 1458-1475.
Gill JS, Pitts K, Rusnak FM, Owen WG, and Windebank AJ (1998). Thrombin
induced inhibition of neurite outgrowth from dorsal root ganglion neurons. Brain
Res 797, 321-327.
Grewal SS, York RD, and Stork PJ (1999). Extracellular-signal-regulated kinase
signalling in neurons. Curr Opin Neurobiol 9, 544-553.
Groth R and Aanonsen L (2002). Spinal brain-derived neurotrophic factor (BDNF)
produces hyperalgesia in normal mice while antisense directed against either
BDNF or trkB, prevent inflammation-induced hyperalgesia. Pain 100, 171-181.
Harrington AM, Brierley SM, Isaacs N, Hughes PA, Castro J, and Blackshaw LA
(2012). Sprouting of colonic afferent central terminals and increased spinal
mitogen-activated protein kinase expression in a mouse model of chronic visceral
hypersensitivity. J Comp Neurol 520, 2241-2255.
Hausdorff WP, Caron MG, and Lefkowitz RJ (1990). Turning off the signal:
desensitization of beta-adrenergic receptor function. FASEB J 4, 2881-2889.
Helke CJ, Krause JE, Mantyh PW, Couture R, and Bannon MJ (1990). Diversity
in mammalian tachykinin peptidergic neurons: multiple peptides, receptors, and
regulatory mechanisms. FASEB J 4, 1606-1615.
Hennebert O, Pelissier MA, Le MS, Wulfert E, and Morfin R (2008). Antiinflammatory effects and changes in prostaglandin patterns induced by 7betahydroxy-epiandrosterone in rats with colitis. J Steroid Biochem Mol Biol 110, 255262.
Henry JL, Krnjevic K, and Morris ME (1975). Substance P and spinal neurones.
Can J Physiol Pharmacol 53, 423-432.
69
Hermanussen S, Do M, and Cabot PJ (2004). Reduction of beta-endorphincontaining immune cells in inflamed paw tissue corresponds with a reduction in
immune-derived antinociception: reversible by donor activated lymphocytes.
Anesth Analg 98, 723-9, table.
Honore P, Kamp EH, Rogers SD, Gebhart GF, and Mantyh PW (2002).
Activation of lamina I spinal cord neurons that express the substance P receptor
in visceral nociception and hyperalgesia. J Pain 3, 3-11.
Hoxie JA, Ahuja M, Belmonte E, Pizarro S, Parton R, and Brass LF (1993).
Internalization and recycling of activated thrombin receptors. J Biol Chem 268,
13756-13763.
Hu J, Zhou J, Li X, Wang F, and Lu H (2011). Schwann cells promote neurite
outgrowth of dorsal root ganglion neurons through secretion of nerve growth
factor. Indian J Exp Biol 49, 177-182.
Hudon V, Sabourin S, Dydensborg AB, Kottis V, Ghazi A, Paquet M, Crosby K,
Pomerleau V, Uetani N, and Pause A (2010). Renal tumour suppressor function
of the Birt-Hogg-Dube syndrome gene product folliculin. J Med Genet 47, 182189.
Ibeakanma C and Vanner S (2010). TNFalpha is a key mediator of the
pronociceptive effects of mucosal supernatant from human ulcerative colitis on
colonic DRG neurons. Gut 59, 612-621.
Jasmin L, Wang H, Tarczy-Hornoch K, Levine JD, and Basbaum AI (1994).
Differential effects of morphine on noxious stimulus-evoked fos-like
immunoreactivity in subpopulations of spinoparabrachial neurons. J Neurosci 14,
7252-7260.
Johansson N, Eriksson P, and Viberg H (2009). Neonatal exposure to PFOS and
PFOA in mice results in changes in proteins which are important for neuronal
growth and synaptogenesis in the developing brain. Toxicol Sci 108, 412-418.
Johnson E, Forland DT, Hetland G, Saetre L, Olstad OK, and Lyberg T (2012).
Effect of AndoSan() on expression of adhesion molecules and production of
reactive oxygen species in human monocytes and granulocytes in vivo. Scand J
Gastroenterol.
70
Jones RC, III, Xu L, and Gebhart GF (2005). The mechanosensitivity of mouse
colon afferent fibers and their sensitization by inflammatory mediators require
transient receptor potential vanilloid 1 and acid-sensing ion channel 3. J Neurosci
25, 10981-10989.
Kang D, Choe C, and Kim D (2005). Thermosensitivity of the two-pore domain
K+ channels TREK-2 and TRAAK. J Physiol 564, 103-116.
Kawabata A, Kawao N, Kitano T, Matsunami M, Satoh R, Ishiki T, Masuko T,
Kanke T, and Saito N (2006). Colonic hyperalgesia triggered by proteinaseactivated receptor-2 in mice: involvement of endogenous bradykinin. Neurosci
Lett 402, 167-172.
Keranen U, Kiviluoto T, Jarvinen H, Back N, Kivilaakso E, and Soinila S (1995).
Changes in substance P-immunoreactive innervation of human colon associated
with ulcerative colitis. Dig Dis Sci 40, 2250-2258.
Kiebles JL, Doerfler B, and Keefer L (2010). Preliminary evidence supporting a
framework of psychological adjustment to inflammatory bowel disease. Inflamm
Bowel Dis 16, 1685-1695.
Kim D (2005). Physiology and pharmacology of two-pore domain potassium
channels. Curr Pharm Des 11 , 2717-2736.
Kimball ES, Prouty SP, Pavlick KP, Wallace NH, Schneider CR, and Hornby PJ
(2007). Stimulation of neuronal receptors, neuropeptides and cytokines during
experimental oil of mustard colitis. Neurogastroenterol Motil 19, 390-400.
King AE, Ackley MA, and Slack JR (1997). Profile of neuronal excitation following
selective activation of the neurokinin-1 receptor in rat deep dorsal horn in vitro.
Brain Res 767, 55-63.
Kobayashi K, Fukuoka T, Obata K, Yamanaka H, Dai Y, Tokunaga A, and
Noguchi K (2005). Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in
rat primary afferent neurons with adelta/c-fibers and colocalization with trk
receptors. J Comp Neurol 493, 596-606.
Kuroki T, Ohta A, Aoki Y, Kawasaki S, Sugimoto N, Ootani H, Tsunada S, Iwakiri
R, and Fujimoto K (2007). Stress maladjustment in the pathoetiology of ulcerative
colitis. J Gastroenterol 42, 522-527.
71
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.
La JH and Gebhart GF (2011). Colitis decreases mechanosensitive K2P channel
expression and function in mouse colon sensory neurons. Am J Physiol
Gastrointest Liver Physiol 301, G165-G174.
Labuz D, Berger S, Mousa SA, Zollner C, Rittner HL, Shaqura MA, SegoviaSilvestre T, Przewlocka B, Stein C, and Machelska H (2006). Peripheral
antinociceptive effects of exogenous and immune cell-derived endomorphins in
prolonged inflammatory pain. J Neurosci 26, 4350-4358.
Labuz D, Schmidt Y, Schreiter A, Rittner HL, Mousa SA, and Machelska H
(2009). Immune cell-derived opioids protect against neuropathic pain in mice. J
Clin Invest 119, 278-286.
Labuz D, Schreiter A, Schmidt Y, Brack A, and Machelska H (2010). T
lymphocytes containing beta-endorphin ameliorate mechanical hypersensitivity
following nerve injury. Brain Behav Immun 24, 1045-1053.
Lapointe TK and Altier C. Mo1859 Visceral Pain Correlates With Neuronal
Outgrowth and Modulation of TRPV1 Trafficking in Dorsal Root Ganglion
Neurons in a Murine Model of Colitis. Gastroenterology 142[5, Supplement 1], S702. 2012. Abstract
Lee CY, Perez FM, Wang W, Guan X, Zhao X, Fisher JL, Guan Y, Sweitzer SM,
Raja SN, and Tao YX (2011). Dynamic temporal and spatial regulation of mu
opioid receptor expression in primary afferent neurons following spinal nerve
injury. Eur J Pain 15, 669-675.
Leo S, D'Hooge R, and Meert T (2010). Exploring the role of nociceptor-specific
sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice.
Behav Brain Res 208, 149-157.
Light AR and Perl ER (1979). Spinal termination of functionally identified primary
afferent neurons with slowly conducting myelinated fibers. J Comp Neurol 186,
133-150.
72
Lomax AE, Linden DR, Mawe GM, and Sharkey KA (2006). Effects of
gastrointestinal inflammation on enteroendocrine cells and enteric neural reflex
circuits. Auton Neurosci 126-127, 250-257.
Longobardi T, Jacobs P, Wu L, and Bernstein CN (2003). Work losses related to
inflammatory bowel disease in Canada: results from a National Population Health
Survey. Am J Gastroenterol 98, 844-849.
Lordal M, Hallgren A, Nylander O, and Hellstrom PM (1996). Tachykinins
increase vascular permeability in the gastrointestinal tract of the rat. Acta Physiol
Scand 156, 489-494.
Lu J, Wang A, Ansari S, Hershberg RM, and McKay DM (2003). Colonic bacterial
superantigens evoke an inflammatory response and exaggerate disease in mice
recovering from colitis. Gastroenterology 125, 1785-1795.
Lundberg LM, Alm P, Wharton J, and Polak JM (1988). Protein gene product 9.5
(PGP 9.5). A new neuronal marker visualizing the whole uterine innervation and
pregnancy-induced and developmental changes in the guinea pig.
Histochemistry 90, 9-17.
Maingret F, Patel AJ, Lesage F, Lazdunski M, and Honore E (1999). Mechanoor acid stimulation, two interactive modes of activation of the TREK-1 potassium
channel. J Biol Chem 274, 26691-26696.
Malan TP, Mata HP, and Porreca F (2002). Spinal GABA(A) and GABA(B)
receptor pharmacology in a rat model of neuropathic pain. Anesthesiology 96,
1161-1167.
Martin-Zanca D, Hughes SH, and Barbacid M (1986). A human oncogene formed
by the fusion of truncated tropomyosin and protein tyrosine kinase sequences.
Nature 319, 743-748.
Mazumdar S and Das KM (1992). Immunocytochemical localization of vasoactive
intestinal peptide and substance P in the colon from normal subjects and patients
with inflammatory bowel disease. Am J Gastroenterol 87, 176-181.
McMahon SB, Armanini MP, Ling LH, and Phillips HS (1994). Expression and
coexpression of Trk receptors in subpopulations of adult primary sensory
neurons projecting to identified peripheral targets. Neuron 12, 1161-1171.
73
McMillian MK, Soltoff SP, and Talamo BR (1987). Rapid desensitization of
substance P- but not carbachol-induced increases in inositol trisphosphate and
intracellular Ca++ in rat parotid acinar cells. Biochem Biophys Res Commun 148,
1017-1024.
Meyer RA, Ringkamp M, Campbell JN, & Raja SN (2006). Peripheral
mechanisms of cutaneous nociception. In Wall and Melzack's Textbook of Pain,
eds. McMahon SB & Koltzenburg M, pp. 3-34. Elsevier/Churchill Livingstone,
London.
Miampamba M and Sharkey KA (1998). Distribution of calcitonin gene-related
peptide, somatostatin, substance P and vasoactive intestinal polypeptide in
experimental colitis in rats. Neurogastroenterol Motil 10, 315-329.
Molliver DC, Wright DE, Leitner ML, Parsadanian AS, Doster K, Wen D, Yan Q,
and Snider WD (1997). IB4-binding DRG neurons switch from NGF to GDNF
dependence in early postnatal life. Neuron 19, 849-861.
Moore KA, Kohno T, Karchewski LA, Scholz J, Baba H, and Woolf CJ (2002).
Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition
in the superficial dorsal horn of the spinal cord. J Neurosci 22, 6724-6731.
Mousa SA, Zhang Q, Sitte N, Ji R, and Stein C (2001). beta-Endorphincontaining memory-cells and mu-opioid receptors undergo transport to peripheral
inflamed tissue. J Neuroimmunol 115, 71-78.
Mousa SA, Machelska H, Schafer M, and Stein C (2002). Immunohistochemical
localization of endomorphin-1 and endomorphin-2 in immune cells and spinal
cord in a model of inflammatory pain. J Neuroimmunol 126, 5-15.
Mousa SA, Shaqura M, Brendl U, Al-Khrasani M, Furst S, and Schafer M (2010).
Involvement of the peripheral sensory and sympathetic nervous system in the
vascular endothelial expression of ICAM-1 and the recruitment of opioidcontaining immune cells to inhibit inflammatory pain. Brain Behav Immun 24,
1310-1323.
Murase K and Randic M (1984). Actions of substance P on rat spinal dorsal horn
neurones. J Physiol 346, 203-217.
74
Neshat S, deVries M, Barajas-Espinosa AR, Skeith L, Chisholm SP, and Lomax
AE (2009). Loss of purinergic vascular regulation in the colon during colitis is
associated with upregulation of CD39. Am J Physiol Gastrointest Liver Physiol
296, G399-G405.
Nguyen C, Coelho AM, Grady E, Compton SJ, Wallace JL, Hollenberg MD,
Cenac N, Garcia-Villar R, Bueno L, Steinhoff M, Bunnett NW, and Vergnolle N
(2003). Colitis induced by proteinase-activated receptor-2 agonists is mediated
by a neurogenic mechanism. Can J Physiol Pharmacol 81, 920-927.
Obara I, Parkitna JR, Korostynski M, Makuch W, Kaminska D, Przewlocka B, and
Przewlocki R (2009). Local peripheral opioid effects and expression of opioid
genes in the spinal cord and dorsal root ganglia in neuropathic and inflammatory
pain. Pain 141, 283-291.
Ochoa-Cortes F, Ramos-Lomas T, Miranda-Morales M, Spreadbury I,
Ibeakanma C, Barajas-Lopez C, and Vanner S (2010). Bacterial cell products
signal to mouse colonic nociceptive dorsal root ganglia neurons. Am J Physiol
Gastrointest Liver Physiol 299, G723-G732.
Page AJ, Brierley SM, Martin CM, Price MP, Symonds E, Butler R, Wemmie JA,
and Blackshaw LA (2005). Different contributions of ASIC channels 1a, 2, and 3
in gastrointestinal mechanosensory function. Gut 54, 1408-1415.
Palecek J, Paleckova V, and Willis WD (2003). Fos expression in spinothalamic
and postsynaptic dorsal column neurons following noxious visceral and
cutaneous stimuli. Pain 104, 249-257.
Palecek J, Paleckova V, and Willis WD (2003). Postsynaptic dorsal column
neurons express NK1 receptors following colon inflammation. Neuroscience 116,
565-572.
Palecek J (2004). The role of dorsal columns pathway in visceral pain. Physiol
Res 53 Suppl 1, S125-S130.
Philippe D, Dubuquoy L, Groux H, Brun V, Chuoi-Mariot MT, Gaveriaux-Ruff C,
Colombel JF, Kieffer BL, and Desreumaux P (2003). Anti-inflammatory properties
of the mu opioid receptor support its use in the treatment of colon inflammation. J
Clin Invest 111, 1329-1338.
75
Phillips HS and Armanini MP (1996). Expression of the trk family of neurotrophin
receptors in developing and adult dorsal root ganglion neurons. Philos Trans R
Soc Lond B Biol Sci 351, 413-416.
Pizzi LT, Weston CM, Goldfarb NI, Moretti D, Cobb N, Howell JB, Infantolino A,
Dimarino AJ, and Cohen S (2006). Impact of chronic conditions on quality of life
in patients with inflammatory bowel disease. Inflamm Bowel Dis 12, 47-52.
Podolsky DK (2002). Inflammatory bowel disease. N Engl J Med 347, 417-429.
Polgar E, Hughes DI, Arham AZ, and Todd AJ (2005). Loss of neurons from
laminas I-III of the spinal dorsal horn is not required for development of tactile
allodynia in the spared nerve injury model of neuropathic pain. J Neurosci 25,
6658-6666.
Ray K (2012). IBS: Distinct neuro-immune patterns defined in IBS subtypes. Nat
Rev Gastroenterol Hepatol.
Ren Y, Zhang L, Lu Y, Yang H, and Westlund KN (2009). Central lateral thalamic
neurons receive noxious visceral mechanical and chemical input in rats. J
Neurophysiol 102, 244-258.
Renganathan M, Cummins TR, and Waxman SG (2001). Contribution of
Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons. J
Neurophysiol 86, 629-640.
Rittner HL, Brack A, Machelska H, Mousa SA, Bauer M, Schafer M, and Stein C
(2001). Opioid peptide-expressing leukocytes: identification, recruitment, and
simultaneously increasing inhibition of inflammatory pain. Anesthesiology 95,
500-508.
Robinson DR, McNaughton PA, Evans ML, and Hicks GA (2004).
Characterization of the primary spinal afferent innervation of the mouse colon
using retrograde labelling. Neurogastroenterol Motil 16, 113-124.
Ross RA (2003). Anandamide and vanilloid TRPV1 receptors. Br J Pharmacol
140, 790-801.
76
Schiechl G, Bauer B, Fuss I, Lang SA, Moser C, Ruemmele P, Rose-John S,
Neurath MF, Geissler EK, Schlitt HJ, Strober W, and Fichtner-Feigl S (2011).
Tumor development in murine ulcerative colitis depends on MyD88 signaling of
colonic F4/80+CD11b(high)Gr1(low) macrophages. J Clin Invest 121, 1692-1708.
Sengupta JN and Gebhart GF (1994). Characterization of mechanosensitive
pelvic nerve afferent fibers innervating the colon of the rat. J Neurophysiol 71,
2046-2060.
Shah Z, Kampfrath T, Deiuliis JA, Zhong J, Pineda C, Ying Z, Xu X, Lu B,
Moffatt-Bruce S, Durairaj R, Sun Q, Mihai G, Maiseyeu A, and Rajagopalan S
(2011). Long-term dipeptidyl-peptidase 4 inhibition reduces atherosclerosis and
inflammation via effects on monocyte recruitment and chemotaxis. Circulation
124, 2338-2349.
Shirai T, Kohara H, and Tabata Y (2012). Inflammation imaging by silica
nanoparticles with antibodies orientedly immobilized. J Drug Target 20, 535-543.
Sivilotti L and Woolf CJ (1994). The contribution of GABAA and glycine receptors
to central sensitization: disinhibition and touch-evoked allodynia in the spinal
cord. J Neurophysiol 72, 169-179.
Steinhoff M, Vergnolle N, Young SH, Tognetto M, Amadesi S, Ennes HS,
Trevisani M, Hollenberg MD, Wallace JL, Caughey GH, Mitchell SE, Williams LM,
Geppetti P, Mayer EA, and Bunnett NW (2000). Agonists of proteinase-activated
receptor 2 induce inflammation by a neurogenic mechanism. Nat Med 6, 151158.
Sugiura Y, Lee CL, and Perl ER (1986). Central projections of identified,
unmyelinated (C) afferent fibers innervating mammalian skin. Science 234, 358361.
Szallasi A, Cortright DN, Blum CA, and Eid SR (2007). The vanilloid receptor
TRPV1: 10 years from channel cloning to antagonist proof-of-concept. Nat Rev
Drug Discov 6, 357-372.
Szigethy E, McLafferty L, and Goyal A (2010). Inflammatory bowel disease. Child
Adolesc Psychiatr Clin N Am 19, 301-18, ix.
77
Talmage EK, Pouliot WA, Schemann M, and Mawe GM (1996). Structure and
chemical coding of human, canine and opossum gallbladder ganglia. Cell Tissue
Res 284, 289-302.
Tornblom H, Lindberg G, Nyberg B, and Veress B (2002). Full-thickness biopsy
of the jejunum reveals inflammation and enteric neuropathy in irritable bowel
syndrome. Gastroenterology 123, 1972-1979.
Tuchscherer MM and Seybold VS (1989). A quantitative study of the coexistence
of peptides in varicosities within the superficial laminae of the dorsal horn of the
rat spinal cord. J Neurosci 9, 195-205.
Valdez-Morales E, Guerrero-Alba R, Ochoa-Cortes F, Benson J, Spreadbury I,
Miranda-Morales M, Lomax A, and Vanner S. Release of endogenous opioids
during a chronic IBD model suppresses the excitability of colonic DRG neurons.
2012. Neurogastroenterol Motil, In Press.
Verma-Gandhu M, Bercik P, Motomura Y, Verdu EF, Khan WI, Blennerhassett
PA, Wang L, El-Sharkawy RT, and Collins SM (2006). CD4+ T-cell modulation of
visceral nociception in mice. Gastroenterology 130, 1721-1728.
Verma-Gandhu M, Verdu EF, Bercik P, Blennerhassett PA, Al-Mutawaly N, Ghia
JE, and Collins SM (2007). Visceral pain perception is determined by the
duration of colitis and associated neuropeptide expression in the mouse. Gut 56,
358-364.
Verma-Gandhu M, Verdu EF, Cohen-Lyons D, and Collins SM (2007).
Lymphocyte-mediated regulation of beta-endorphin in the myenteric plexus. Am J
Physiol Gastrointest Liver Physiol 292, G344-G348.
Waldmann R, Champigny G, Bassilana F, Heurteaux C, and Lazdunski M (1997).
A proton-gated cation channel involved in acid-sensing. Nature 386, 173-177.
Watanabe T, Kubota Y, and Muto T (1997). Substance P containing nerve fibers
in rectal mucosa of ulcerative colitis. Dis Colon Rectum 40, 718-725.
Weigmann B, Tubbe I, Seidel D, Nicolaev A, Becker C, and Neurath MF (2007).
Isolation and subsequent analysis of murine lamina propria mononuclear cells
from colonic tissue. Nat Protoc 2, 2307-2311.
78
Wells DG, Talmage EK, and Mawe GM (1995). Immunohistochemical
identification of neurons in ganglia of the guinea pig sphincter of Oddi. J Comp
Neurol 352, 106-116.
Wiedermann CJ, Sacerdote P, Propst A, Propst T, Judmaier G, Kathrein H,
Vogel W, and Panerai AE (1994). Decreased beta-endorphin content in
peripheral blood mononuclear leukocytes from patients with Crohn's disease.
Brain Behav Immun 8, 261-269.
Woolf CJ and Ma Q (2007). Nociceptors--noxious stimulus detectors. Neuron 55,
353-364.
Wouters MM, Van WS, Casteels C, Nemethova A, de VA, Van OL, Van den
Wijngaard RM, Van LK, and Boeckxstaens G (2012). Altered brain activation to
colorectal distention in visceral hypersensitive maternal-separated rats.
Neurogastroenterol Motil 24, 678-e297.
Xiong ZG, Zhu XM, Chu XP, Minami M, Hey J, Wei WL, MacDonald JF, Wemmie
JA, Price MP, Welsh MJ, and Simon RP (2004). Neuroprotection in ischemia:
blocking calcium-permeable acid-sensing ion channels. Cell 118, 687-698.
Xu Y, Qian L, Zong G, Ma K, Zhu X, Zhang H, Li N, Yang Q, Bai H, Ben J, Li X,
Xu Y, and Chen Q (2012). Class A scavenger receptor promotes cerebral
ischemic injury by pivoting microglia/macrophage polarization. Neuroscience
218, 35-48.
Yamamoto H, Morise K, Kusugami K, Furusawa A, Konagaya T, Nishio Y,
Kaneko H, Uchida K, Nagai H, Mitsuma T, and Nagura H (1996). Abnormal
neuropeptide concentration in rectal mucosa of patients with inflammatory bowel
disease. J Gastroenterol 31, 525-532.
Yamdeu RS, Shaqura M, Mousa SA, Schafer M, and Droese J (2011). p38
Mitogen-activated protein kinase activation by nerve growth factor in primary
sensory neurons upregulates mu-opioid receptors to enhance opioid
responsiveness toward better pain control. Anesthesiology 114, 150-161.
Yermolaieva O, Leonard AS, Schnizler MK, Abboud FM, and Welsh MJ (2004).
Extracellular acidosis increases neuronal cell calcium by activating acid-sensing
ion channel 1a. Proc Natl Acad Sci U S A 101, 6752-6757.
79
Yiangou Y, Facer P, Smith JA, Sangameswaran L, Eglen R, Birch R, Knowles C,
Williams N, and Anand P (2001). Increased acid-sensing ion channel ASIC-3 in
inflamed human intestine. Eur J Gastroenterol Hepatol 13, 891-896.
Zimmermann K, Leffler A, Babes A, Cendan CM, Carr RW, Kobayashi J, Nau C,
Wood JN, and Reeh PW (2007). Sensory neuron sodium channel Nav1.8 is
essential for pain at low temperatures. Nature 447, 855-858.
80