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Theses and Dissertations--Physiology
Physiology
2014
ATTENUATING TRIGEMINAL
NEUROPATHIC PAIN BY REPURPOSING
PIOGLITAZONE AND D-CYCLOSERINE IN
THE NOVEL TRIGEMINAL
INFLAMMATORY COMPRESSION MOUSE
MODEL
Danielle N. Lyons
University of Kentucky, [email protected]
Recommended Citation
Lyons, Danielle N., "ATTENUATING TRIGEMINAL NEUROPATHIC PAIN BY REPURPOSING PIOGLITAZONE AND DCYCLOSERINE IN THE NOVEL TRIGEMINAL INFLAMMATORY COMPRESSION MOUSE MODEL" (2014). Theses and
Dissertations--Physiology. Paper 19.
http://uknowledge.uky.edu/physiology_etds/19
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Danielle N. Lyons, Student
Dr. Karin N. Westlund High, Major Professor
Dr. Bret Smith, Director of Graduate Studies
ATTENUATING TRIGEMINAL NEUROPATHIC PAIN BY REPURPOSING
PIOGLITAZONE AND D-CYCLOSERINE IN THE NOVEL TRIGEMINAL
INFLAMMATORY COMPRESSION MOUSE MODEL
DISSERTATION
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in the College of Medicine at the University of Kentucky
By
Danielle Nicole Lyons
Lexington, Kentucky, United States of America
Director: Dr. Karin N. Westlund High
Lexington, KY
2014
Copyright © Danielle N. Lyons 2014
ABSTRACT OF DISSERTATION
ATTENUATING TRIGEMINAL NEUROPATHIC PAIN BY REPURPOSING
PIOGLITAZONE AND D-CYCLOSERINE IN THE NOVEL TRIGEMINAL
INFLAMMATORY COMPRESSION MOUSE MODEL
Approximately 22% of the United States population suffers from a chronic
orofacial pain condition. One such condition is known as trigeminal neuropathic
pain frequently reported as continuous aching and burning pain, often
accompanied by intermittent electrical shock-like sensations. Dental procedures
or trauma are known causes of peripheral trigeminal nerve injury and
inflammation. Patients who have this type of facial pain also suffer from
emotional distress. For these reasons, trigeminal neuropathic pain needs to be
studied in more detail to improve the understanding of the etiology and
maintenance of this condition, as well as to develop effective treatment
strategies. The first experiment was focused on characterizing the behavioral
aspects of the Trigeminal Inflammatory Compression (TIC) mouse model. The
findings determined that the TIC injury model induced mechanical and cold
hypersensitivity that persist at least 21 weeks. This orofacial, neuropathic pain
condition was accompanied by anxiety- and depressive-like behaviors at week 8
post injury. The TIC injury mouse model’s chronicity and development of
psychosocial impairments demonstrated its usefulness as a facial pain model.
The second experiment used the mouse TIC injury model to test the ability of
pioglitazone (PIO), a PPARγ agonist used clinically for treatment of diabetes, on
alleviating trigeminal pain. A single low dose of PIO had no effect, but a higher
dose attenuated facial pain. The third experiment determined that combining
ineffective low doses of PIO and D-cycloserine (DCS) produced a potentiated
anti-allodynic response of these drugs and attenuated the anxiety associated
with the TIC injury. Ex vivo studies revealed that cortical mitochondrial
dysfunction occurred after the TIC injury but could be reversed by the
combination of DCS/PIO which improves mitochondrial function. Overall, the
present studies determined that the novel mouse TIC injury model is a clinically
relevant facial neuropathic pain model. The results suggest that PPARγ and
brain mitochondria may represent new molecular targets for the treatment of
trigeminal neuropathic pain. These studies support the future “repurposing” of
PIO and DCS as well as the combination of the two drugs for this new use in
patients with trigeminal neuropathic pain.
KEYWORDS: trigeminal inflammatory compression, mouse model,
PPARγ, D-cycloserine, mitochondria
Danielle N. Lyons
December 3, 2014
ATTENUATING TRIGEMINAL NEUROPATHIC PAIN BY REPURPOSING
PIOGLITAZONE AND D-CYCLOSERINE IN THE NOVEL TRIGEMINAL
INFLAMMATORY COMPRESSION MOUSE MODEL
By
Danielle Nicole Lyons
Dr. Karin N. Westlund High
Director of Dissertation
Dr. Bret N. Smith
Director of Graduate Studies
This dissertation is dedicated to my Rock and my Redeemer, my Lord and
my Savior, Jesus Christ...
“Let all that I am praise the Lord; may I never forget the good things He
does for me.”- Psalm 103:2
ACKNOWLEDGEMENTS
First of all, I have to thank my mentor, Dr. Karin Westlund High. Thank you for
the funding that you have provided me for this project. Thank you also for
allowing me freedom to research what I wanted. You trusted me and believed in
my vision, so thank you. I admire your courage in life and respect the passion
you have for research. I cannot thank you enough for all the time you have
invested into me. I will never forget all of your encouragement and support
through my comprehensive exams as well as this dissertation process.
Secondly, I have to thank everyone in the High lab that has been a great support
to me. Liping, thank you for teaching me proper pharmacology and animal
behavioral methods. Thank you also so much for the suggestions/revisions to
this dissertation and the many times you have helped me with my experiments.
You have been my constant companion and dear friend through this entire
process. I thank you so much for the guidance, loyalty, and listening ear you
have provided. Fei, thank you for teaching me your wonderful surgery techniques
on the mice and rats. Thank you so much for all your support and suggestions on
many other projects. Your input and smile has always made my day easier. Bob,
thank you so much for teaching me how to immunostain and having a wonderful
gracious attitude. Sabrina, thank you for your many suggestions and useful
input. Tracey, you are and forever will be my “partner in crime.” From testing all
day on weekends to singing in the stairwell, our therapy sessions truly have been
some of the best memories I have had in graduate school. And to all of the past
of rotating lab members and students, thank you for any help you provided.
I would also like to thank my committee members. Dr. Taylor, thank you for
allowing me to rotate in your lab year one. I learned so much from that 8 week
rotation. Thank you for always challenging me and pushing me to be the best
that I can be. Dr. Frolenkov, thank you for your constant belief in me and my
abilities. Our scientific and non-scientific conversations have been such an
encouragement. Dr. Gerhardt, thank you so much for all of the knowledge you
have provided about current literature and neuroscience. Your ability to keep me
focused has helped tremendously along with your positive and diplomatic
attitude. Dr. De Leeuw, thank you so much for allowing me to sit in on the
Orofacial Pain Seminars and all of the input you have provided from the clinical
perspective of orofacial pain. You all have been amazing in availability to me
and for that, I cannot thank you enough.
I would like to extend a BIG THANK YOU to the department of Physiology as well
as the University of Kentucky for funding me my first two years. Thank you so
much to my fellow grad students and friends who have been there to enjoy this
journey with me. Thank you so much Sherry and Cindy, my “big sisters” who
have made this department so much fun and have been there to celebrate every
moment with me.
Thank you to Drs. Frolenkov and Campbell for the
Communication Skills workshop and PGY 774. Your investment in students is
admirable and appreciated. Thank you to Drs. Randall and Speck for being
iii
amazing teaching mentors. I have learned so much from the both of you and
love you dearly. Thank you to Dr. Saatman for all your support during PGY 602your kindness will never be forgotten. Thank you to all of the faculty and
professors who have invested their time to teach me physiology. When I think of
you all, it will be fondly.
Thank you so much to the Taylor lab. Renee and Suzanne, you both have been
my partners in crime and I will never forget you. Thank you also to the Sullivan
lab: Drs. Sullivan, Pandya, and Patel for their time and mentoring.
Thank you so much to COBRE grant under Dr. Jeff Ebersole that supported
some of the funding for this project. I want to thank Drs. Danaher, Miller, and
Carlson for all of your valued input and direction with these experiments. Thank
you for your revisions/editions on several papers and for all the time you each
invested. Robert, thank you for always being willing to order what I needed.
Your joy is remarkable and came just when I needed it so thank you. Dr. Miller,
thank you for wise criticisms and positive comments. You always pushed me to
better explain myself- a tool that will no doubt help me in the future
Thank you to my family. You guys have seen me through every up and every
down. You always have my back and never withdrawal your love. You guys
remind me of the verse in Proverbs 31 when it states, “you are worth more than
rubies.” God overflowed my cup when He gave me all of you. Thank you to my
“fave”, Cameron. I love you neph/bro! Any time spent with you made me feel
blessed. Kenna, my lovely sister, thank you for all you give. You love is awestriking and you are the best big sister a girl could ask for. Connor, thank you for
bringing life to our family. Kerry, you will always be my favorite brother-in-law.
To my parents, thank you for always being a phone call away. Thank you for
always agreeing with even when if I was wrong. To my wonderful mother and
best friend, you are the inspiration of this dissertation. Any time I wanted to give
up, it was the thought of you that would keep me going. For you were right when
you said, “Anything great, is worth fighting for.” And you, Mommy, are definitely
worth fighting for. Thank you to my steadfast father. You reflect the Father’s love
to me in such a pure way. Thank you for our many golf outings and dinners that
reset my battery. Thank you so much for never getting tired of me “talking
science.” I love you both so much.
Thank you to all my friends who have been there to pray with me and listen to
me. Thank you for your constant support through this process. There are too
many to name, but I love you all dearly.
Lastly, thanks be to God. Who gave me rest when I needed it, and strength
when I could not go on. “To our God and Father be glory for ever and ever.
Amen.” -Philippians 4:20
iv
TABLE OF CONTENTS
Acknowledgements
List of Tables
List of Figures
iii
ix
x
1 Chapter 1: Introduction and Background
1.1
Introduction of Continuous Trigeminal Neuropathic Pain
1.2
Anatomy of the Trigeminal System
1.3
Biopsychosocial Model of Pain
1.4
Current Treatments
1.5
Trigeminal Inflammatory Compression (TIC) Injury
1.6
Scope and Hypothesis of the Dissertation
1
5
8
9
11
13
2 Chapter 2: Induced Facial Pain, Anxiety-and Depression Related
Behaviors Associated with Trigeminal Inflammatory Compression
(TIC) Injury
2.1 Introduction
23
2.2 Materials and Methods
26
2.2.1 Animals
26
2.2.2 Trigeminal Inflammatory Compression (TIC) Injury Surgery 26
2.2.3 Behavioral Tests
27
2.2.3.1 Assessment of Mechanical Allodynia
28
2.2.3.2 Assessment of Thermal Hypersensitivity
29
2.2.3.3 Acoustic Startle Disturbance
30
2.2.3.4 Two Compartment Light-Dark Box Preference Test 31
2.2.3.5 Open Field Exploratory Activities
32
2.2.3.6 Elevated Plus Maze Task
33
2.2.4 Statistical Analysis
34
2.3 Results
34
2.3.1 Animals with TIC Injury Displayed Unilateral Whisker Pads
Mechanical Allodynia
34
2.3.2 TIC Injury Induced a Unilateral Cold Allodynia of the V2
Area
36
2.3.3 Animals with TIC Injury Displayed Anxiety- and
Depressive-Like Behaviors in Two Compartment Light-Dark
Preference Testing
37
2.3.4 Acoustic Disturbance Affected Mice with TIC in the Elevated
Plus Maze Task
38
2.3.5 Mice with TIC Injury Showed Less Exploratory Activities
39
2.4 Discussion
39
2.4.1 TIC Injury Model Mimics Clinical Facial Neuropathic Pain
39
2.4.2 TIC Injury Developed Anxiety- and Depressive -Like Behaviors
Similar to Patients Suffering with Chronic Facial Pain
41
v
2.4.3 Decreased Fear Response in Mice with TIC after Acoustic
Disturbance
2.5 Conclusion
3 Chapter 3: Rapid Effects of PPAR-Agonists on Alleviation of
Trigeminal Pain
3.1 Introduction
3.2 Materials and Methods
3.2.1 Animals
3.2.2 Trigeminal Inflammatory Compression (TIC) Surgery
3.2.3 Assessment of Mechanical Allodynia on the Mouse Whisker
Pad
3.2.4 Immunohistological Study
3.2.5 Image Analysis
3.2.6 Drug Preparation and Administration
3.2.7 Statistical Analysis
3.3 Results
3.3.1 Mice with TIC Injury Demonstrated Unilateral Whisker Pad
Mechanical Allodynia
3.3.2 The Effect of PPAR Agonists on Mechanical Allodynia of
Mice with TIC Injury
3.3.2.1 PPARγ Agonist, Pioglitazone, Attenuated Mechanical
Allodynia in Mice with TIC Injury
3.3.2.2 PPARβ/δ Agonist Moderately Attenuated Mechanical
Allodynia in the Mice with TIC Injury
3.3.2.3 PPARα Agonist Had No Effect on the Mice with TIC
Injury
3.3.2.4 PPARγ Antagonist, GW9662, Blocked Analgesic
Effects of Pioglitazone
3.3.3 PPARγ Was More Immunoreactive in the TBSNC of Mice
with TIC injury
3.4 Discussion
3.5 Conclusion
4 Chapter 4: Low-Dose Combination of Pioglitazone and D-Cycloserine
Attenuated Orofacial Pain by Improving Mitochondrial Dysfunction
4.1 Introduction
4.2 Materials and Methods
4.2.1 Animals
4.2.2 Trigeminal Inflammatory Compression (TIC) Injury
4.2.3 Behavioral Assays
4.2.3.1 Detecting Mechanical Allodynia with von Frey Fiber
Test
vi
44
47
57
60
60
60
60
60
62
62
64
65
65
66
66
67
67
68
68
70
74
83
88
88
88
88
88
4.2.3.2 Light-Dark Box Preference Testing
4.2.4 Drug Preparation and Administration
4.2.4.1 Drug Preparation
4.2.4.2 Drug Administration
4.2.4.2.1 Experiment 1: Treatment of Mechanical
Allodynia with NMDA Receptor Agonist/
Antagonist, DCS
4.2.4.2.2 Experiment 2: One-time Injection of
Combination of DCS/PIO
4.2.4.2.3 Experiment 3: 7-day Treatment of 80 mg/kg
Dose of DCS Followed by a Bolus of
100 mg/kg Dose of PIO on the 7th Day
4.2.4.2.4 Experiment 4: Single Dose of Mitochondrial
Uncoupler 2,4-DNP
4.2.5 Mitochondrial Isolation Assays
4.2.5.1 Isolated Mitochondria Preparation
4.2.5.2 Bioscience Seahorse XFe24 Flux Analyzer Assay
4.2.6 Statistical Analysis
4.3 Results
4.3.1 Unilateral Facial Mechanical Allodynia in Mice with TIC
Injury
4.3.2 D-cycloserine Attenuated Mechanical Allodynia But Did
Not Relieve Anxiety Behaviors Associated with
Hypersensitivity
4.3.3 Attenuation of Mechanical Alloydnia and Anxiety by
Combination of Ineffective Low Doses of D-cycloserine
and Pioglitazone
4.3.4 2.4-DNP Attenuated Mechanical Allodynia in Mice with TIC
Injury
4.3.5 Cortex Mitochondria Had a Decreased Respiratory
Control Ratio in Mice with TIC Indicating Cortical
Mitochondrial Dysfunction
4.3.6 Drug Combination Treatment Increased Respiratory
Control Ratio in Brainstem Mitochondria of Mice with
TIC Injury
4.4 Discussion
4.5 Conclusion
5 Chapter 5: Overall Discussion and Conclusions
5.1 Trigeminal Inflammatory Compression (TIC) Injury is a Clinically
Relevant Orofacial Neuropathic Pain Model
5.2 PPARγ is an Important Molecular Target for the Treatment
of Trigeminal Neuropathic Pain
5.3 Mitochondrial Dysfunction is One Underlying Mechanism of
Trigeminal Neuropathic pain
5.4 Limitations and Future Studies
vii
89
89
89
89
90
91
91
92
92
92
93
95
96
96
97
98
100
100
102
102
108
119
121
123
124
5.5 Significance and Innovation
5.6 Conclusion
130
134
Appendices
Appendix 1. Abbreviations
135
References
138
Vita
155
viii
LIST OF TABLES
1.1 Chronic Pain Is More Prevalent Than Other Major Diseases
2.1 Light-Dark Box Data
2.2 Elevated Plus Maze Data
ix
16
48
49
LIST OF FIGURES
1.1 Anatomy of Trigeminal Nerve
17
1.2 Trigeminal Brainstem Sensory Nuclear Complex (TBSNC) with
Specific Nociceptor Fiber Representation
18
1.3 Functional Magnetic Resonance Imaging (fMRI) of a Patient
Suffering from Trigeminal Neuropathic Pain
19
1.4 The Matrix of “Pain Sensation” and “Pain Affect.”
20
1.5 The Trigeminal Sensory System in Rodents
21
1.6 The CCI-ION Injury Model in Rats Compared to the TIC Injury Model in
Mice
22
2.1 TIC Injury Induced Mechanical and Cold Allodynia
50
2.2 Mice with TIC Injury Had Anxiety- and Depressive-Like Behavior in the
Light-Dark Box
52
2.3 Acoustic Disturbance Affected Mice with TIC Injury in Elevated
Plus Maze
53
2.4 Mice with TIC Injury Showed Decreased Exploratory Activity
in Open Field
55
3.1 Mice with TIC Injury Developed Unilateral Mechanical Allodynia on
the Ipsilateral Whisker Pad
75
3.2 Pioglitazone (PIO) Attenuated Mechanical Allodynia in the Mice
with TIC Injury
76
3.3 PPARγ Immunoreactivity Increased in TBSNC of the Mice with TIC
Injury
78
3.4 PPARγ Immunoreactivity Localized Throughout the Trigeminal
Brainstem Sensory Nuclear Complex in Mice with TIC Injury
80
3.5 PPARγ Immunoreactivity at the Spinal Trigeminal Nucleus
Interpolaris in Sham Mice vs. Mice with TIC Injury
82
4.1 TIC Injury Induced Unilateral Whisker Pad Mechanical Allodynia
109
4.2 DCS Attenuated Mechanical Allodynia, but Does Not Reverse
Anxiety-Like Behavior in the Mice with TIC Injury
110
4.3 The Combination of DCS and PIO Attenuated Mechanical Allodynia
and Anxiety-Like Behavior in the Mice with TIC Injury
112
4.4 A Mild Mitochondrial Uncoupler Attenuated the Mechanical Allodynia
in the Mice with TIC Injury
114
4.5 Isolated Cortical Mitochondria from the Mice with TIC Injury Showed an
Increased State III and State IV OCR, but Decreased RCR which is
Reversed with the Drug Combination of DCS + PIO
115
4.6 The Drug Combination (DCS +PIO) Increased the Brainstem
Mitochondrial RCR in the Mice with TIC Injury
117
x
CHAPTER ONE
INTRODUCTION AND BACKGROUND
1.1 . Introduction of Continuous Trigeminal Neuropathic Pain
Aristotle believed that pain along with all other sensations e.g. vision/taste
were perceived in the heart (Chen 2011; Rey 1993). For a culture in the prime of
the dramatic theatre, where masks were used to describe an actor’s emotion,
perhaps Aristotle and his fellow philosophers at the time thought that “emotions”
were revealed in face, but felt in the heart. However, in the 1500s, autopsies
performed by Andreas Vesalius supported the theory that the brain was in fact
the site of perception of all sensations including pain. In the 1600s, René
Descartes, the “Father of Modern Philosophy,” reasoned that peripheral sensory
nerve fibers carried the “pain” signal to the spinal cord. The signal was then
thought to be transmitted to the brain along neurons and then to the pineal organ
(Descartes’ believed site of pain perception) (Chen 2011; Rey 1993). In 1664,
Thomas Willis published the Cerebri Anatome which reinforced the brain as the
site of pain and the cerebral cortex as the key player in pain perception. Since his
discovery, countless scientists have added to the anatomy and theory of pain
perception in hopes of answering two major questions: 1) What is pain? and 2)
How can pain be stopped?
Although these questions are still being pursued, previous studies have allowed
the International Study of Pain (IASP) to form a solid definition:
“Pain is an unpleasant sensory and emotional
experience associated with actual or potential
1
tissue damage, or described in terms of such
damage.” (IASP 2014)
However, this definition barely begins to describe the mechanisms of pain or the
many different types of pain that exist. For example, allodynia is specific “pain
due to stimulus that does not normally provoke pain” while hyperalgesia is
“increased pain from a stimulus that normally provokes pain” (IASP 2014). The
American Academy of Pain Medicine provides a good definition of chronic pain,
“Pain that persists due to pain signals
continuing to fire in the nervous system for
weeks, months, even years (pain resulting from
damage to the peripheral nerves or central
nervous system.” (AAPM, 2014)
Generally, pain is considered chronic if it lasts longer than 3 months.
Surprisingly, 100 million American citizens suffer from some form of chronic pain
(AAPM 2014). Table 1.1 shows that patients with chronic pain are four times
more prevalent in the population than patients with diabetes as well as patients
suffering from heart disease/stroke (AAPM 2014). These statistics are most likely
due to the fact that in every disease “damage” occurs in the infected tissue often
times eliciting a painful response. Hence if a person has a chronic disease, then
they could also suffer from chronic pain. Due to the numerous chronic diseases,
there are multiple classifications of chronic pain including chronic neuropathic
pain. According to IASP, neuropathic pain is defined as
“Pain caused by a lesion or disease of the
somatosensory nervous system.
2
Note: Neuropathic pain is a clinical description
(and not a diagnosis) which requires a
demonstrable lesion or a disease that satisfies
established neurological diagnostic criteria. The
term lesion is commonly used when diagnostic
investigations (e.g. imaging, neurophysiology,
biopsies and laboratory tests) reveal an
abnormality or when there was obvious trauma.
The term disease is commonly used when the
underlying cause of the lesion is known (e.g.
stroke, vasculitis, DIABETES MELLITUS,
genetic abnormality). Somatosensory refers to
information about the body per se including
visceral organs, rather than information about
the external world (e.g., vision, hearing, or
olfaction). The presence of symptoms or signs
(e.g., touch-evoked pain) alone does not justify
the use of the term neuropathic. Some disease
entities, such as TRIGEMINAL NEURALGIA,
are currently defined by their clinical
presentation rather than by objective diagnostic
testing. Other diagnoses such as postherpetic
neuralgia are normally based upon the history.
It is common when investigating neuropathic
pain that diagnostic testing may yield
inconclusive or even inconsistent data. In such
instances, clinical judgment is required to
reduce the totality of findings in a patient into
one putative diagnosis or concise group of
diagnoses.” (IASP 2014)
The note in the IASP definition describes neuropathic pain as a “clinical
description” because of the difficulties in diagnosing patients with neuropathic
pain when no cause of the pain is currently known or observed. Interestingly
enough, at least half of the note description was referring to orofacial pain
defined as “pain and dysfunction affecting motor and sensory transmission in the
trigeminal nerve system” (De Leeuw 2008).
3
Trigeminal neuropathic pain was first alluded to by Areataues of
Cappadocia in the 2nd Century. He not only described migraines, but also
described “facial spasms and distortions of the countenance” (Nurmikko &
Eldridge 2001). In 1756, Nicolaus André coined the term “tic douloureux,”
meaning painful twitch, to describe these facial spasms currently known today as
Trigeminal Neuralgia. However, it was not until 1773 when a full account of
trigeminal neuralgia was published by John Fothergill. Although trigeminal
neuralgia is the most well-known of the trigeminal neuropathic pain conditions,
modern day diagnosis has helped better classify the different types of trigeminal
neuropathic pain in order to determine proper treatment.
Zakrzweska and colleagues (2013) depicts the most recent classifications
of chronic neuropathic facial disorders using the following features to categorize
the pain : 1) location, 2) timing, 3) quality severity, 4) aggravating factors, 5)
associated factors, 6) examination 7) investigations (scans or sensory testing),
and 8) pain management (Zakrzewska 2013). Trigeminal neuralgia, episodic
chronic pain, is viewed separately from continuous trigeminal neuropathic pain
that exists in the absence of the paroxysmal attacks. For clinicians, continuous
trigeminal neuropathic pain can often times be the most difficult to treat because
of the amount of peripheral and central factors that contribute to the exact cause
of the pain (Blasberg & Greenberg 2008; De Leeuw 2008; Okeson 2005;
Zakrzewska 2013). Even though the etiology of every syndrome cannot be
defined, understanding the anatomy of the trigeminal system is certainly helpful
in determining some problems with trigeminal pain transmission.
4
1.2 . Anatomy of the Trigeminal System
The trigeminal nerve is the fifth cranial containing both a motor and
sensory nuclei relaying information to and from the orofacial region. This largest
cranial nerve consists of three main peripheral branches: ophthalmic (V 1), the
maxillary (V2), and the mandibular (V3). Figure 1.1 (De Leeuw 2008) depicts the
specific orofacial region innervated by each main branch of the trigeminal nerve.
The ophthalmic branch innervates the region comprised of the forehead and the
upper 1/3 of the face, including the meninges. The maxillary innervation region is
the middle 1/3 of the face below the eye encompassing the sinuses and the
mouth region. The mandibular branch innervates the lower parts of mouth,
mandible bone with the temporomandibular joint, and ear (De Leeuw 2008).
The relay of neuronal transmission from the periphery for central
perception begins with a stimulus activating the peripheral pseudo-unipolar
sensory neurons. Noxious input is transduced by the primary afferent
nociceptors. Nociceptors are located in the skin, viscera, joints, vasculature, and
muscle, although they are typically silent in deep structures. With specificity in
function, modality, and density, nociceptors are able to respond to any noxious
heat, cold, mechanical, and chemical stimulus. The primary nociceptor subtypes
relay their information via primary afferents which include: A-delta afferents,
thinly myelinated fibers with a conduction velocity of > 2 m/s and C-fiber
afferents, unmyelinated fibers with a slower conduction velocity <2 m/s (Wall &
Melzack 1999; Westlund & Willis 2015; Willis & Westlund 1997). These two
primary afferent axonal subtypes respond similarly to the nociceptive stimuli with
5
the exception that the A-delta fibers surpass the C fibers in firing rate and do not
respond to chemical stimuli (Voscopoulos & Lema 2010; Wall & Melzack 1999).
As compared to A-beta fibers, primary afferents that respond to low threshold
mechanical stimuli, the C and A-delta nociceptors have a significantly higher
activation threshold in order to respond with an action potential in “normal
conditions.” Therefore, slight touch (a low threshold stimulus) across the face
elicits firing in the A-beta fibers while a slap across the face (a mix of low and
high threshold stimuli) elicits the firing of not only the A-beta fibers, but also the C
and A-delta fibers to generate action potentials.
These primary afferent neurons have their cell bodies in the peripheral
trigeminal ganglion also known as the Gasserian ganglion, a collection of ganglia
from all three branches of the trigeminal system. Together, the peripheral
neurons enter the trigeminal dorsal root entry zone and may ascend/descend the
brainstem to synapse at different levels of the trigeminal brainstem sensory
nuclear complex (TBSNC) depicted in Figure 1 and 2. The nuclei composing the
TBSNC are the main sensory nucleus and the spinal trigeminal nucleus (sp5),
which is composed of three subnuclei: oralis, interpolaris, and caudalis.
Figure1.2 (DaSilva & DosSantos 2012) shows the distribution of the A and C
fibers. The A-beta fibers synapse primarily in the main sensory nucleus located in
the pons and some in the sp5 oralis. The A-delta and C-fibers synapse onto
second order neurons in the sp5 oralis, interporalis and sp5 caudalis, with the
majority of the C-fibers synapsing in sp5 caudalis.
6
The second order neurons in the TBSCN ascend parallel with the medial
lemniscus pathway, the sensory fibers arising from spinal levels. They project
from the brainstem and synapse onto neurons in the ventroposteromedial (VPM)
nucleus of the thalamus, “the sensory relay center,” forming what is known as the
trigeminothalamic pathway. From the VPM the third order neurons in the
thalamus will synapse onto postcentral gyrus neurons (somatosensory cortex),
the actual site of pain perception. Figure 1.3 (Becerra et al 2006) depicts this
pathway using fMRI BOLD imaging from a patient suffering from trigeminal
neuropathic pain. When a specific noxious stimulus was applied to the facial
region, all areas of the trigeminal system were highlighted implicating
involvement of the entire system in chronic facial pain.
However, the neurological system is extremely complicated involving
many signaling pathways. The thalamus has received the title “the sensory relay
center” because it not only receives input from the spinal trigeminal nucleus and
then relays it to the cortex, but also receives input from all levels of the brainstem
and the spinal cord and delivers their messages to the higher order areas of the
brain. Figure1.4 (May 2009) illustrates the “pain matrix “thereby highlighting all
the areas of the brain influencing pain perception (i.e. primary and secondary
somatosensory cortex, insula cortex, prefrontal cortex, cingulate cortex, posterior
parietal cortex, amygdala, thalamus, periaqueductal gray, basal ganglia,
supplementary motor area, and the nucleus accumbens (not shown in the
figure)). This would indicate and support the idea that pain processes are not
7
only just a response to a peripheral nociceptive input, but are also activation of a
central network.
1.3 . Biopsychosocial Model of Pain
Wall and Melzack (1999) highlighted this feature of pain when they said
pain can best be described in two aspects: “pain sensation” and “pain affect.”
This is referring not only to the biological disturbance and emotional distress that
a chronic pain patient experiences, but also including the disruptions in
psychosocial functioning associated with this condition/syndrome (Engel 1997;
Quintner et al 2008; Wall & Melzack 1999).
Perhaps Aristotle’s definition of pain perception occurring in the heart was
not exactly wrong. Although physiologically incorrect, Aristotle understood the
emotional aspect of pain that the philosophers in the 17th century overlooked.
“Pain” does not exist in a vacuum and cannot exclusively be caused by one thing
such as the historical biomedical model of pain suggests. This model illustrates a
direct cause to the pain that a person perceives. It illustrates pain only as a
symptom of an injury that occurred elsewhere in the body (Blasberg & Greenberg
2008; De Leeuw 2008). However, not every painful syndrome appears to have a
direct cause. Therefore, pain cannot solely be due to “sensation.” In 1977,
George Engel coined the term “biopsychosocial model of illness” in which he
challenged the biomedical model of “cause-and-effect” and begin describing
illness as the interaction of the biological, psychological, social, and cultural
components that are exposed to a person (Engel 1977). All of these factors play
8
a key role in a person’s perception and self-diagnosis of their pain (Quintner et al
2008).
Anxiety related to trigeminal neuropathic pain was first described by an
Arab physician, Jujani, in the 11th century (Nurmikko & Eldridge 2001). Today,
many patients with chronic trigeminal neuropathic pain also report numerous
comorbid physical and psychological conditions as well as psychosocial and
economic issues (i.e. medical expenses, loss of work, sleep disturbances,
anxiety, depression, and family conflict) (Asmundson & Katz 2009; Burris et al
2010; De Leeuw 2008; Nicholson & Verma 2004; Porto F 2011). Besides the
anxiety and depression that are related to pain, some patients may often even
experience cognitive impairment due to their pain. ((Ji et al 2010). Several
studies have supported the amygdala, nucleus accumbens, and pre-frontal
cortex, as major players in the observed emotional or cognitive effects (Apkarian
2004; Apkarian et al 2004a; Apkarian et al 2004b; Ji et al 2010) . However, with
the pain matrix being so interconnected, often times it is difficult to determine
which higher order brain regions are solely responsible for the changes observed
in the brain. Therefore, this makes treating the patients with chronic continuous
trigeminal pain very difficult.
1.4.
Current Treatments
Clinicians have progressed in their ability to provide treatment for patients
suffering from chronic orofacial pain. They are beginning to understand the
comorbidities of this syndrome and therefore treat not only the “sensation,” but
9
also the “pain affect.” Pharmacological approaches along with psychological
therapy have been implemented.
The first line of pharmacological treatment is purely based on the pain
assessment and history of that patient. If local anesthetics fail to relieve pain,
many studies show that tricyclic antidepressants such as amitriptyline, doxepin,
or nortriptyline improve the pain of patients as well as their anxiety/depression
related to their pain. Anticonvulsants, such as gabapentin, are typically not
effective in subduing their pain (De Leeuw 2008). Still, pregabalin and tramadol
have also been prescribed. However, many patients still struggle to find any
medications that attenuate their pain.
To treat the “pain affect,” clinicians and therapist often adapt the
biobehavioral model of therapy in order to treat patients suffering with chronic
pain. Carlson (2008) states the biobehavioral approach as “integrating the
important roles biological factors play in governing human function with the
influences of behavioral factors” (Carlson 2008). Cognitive behavioral therapy
(CBT) is one type of biobehavioral therapy that focuses on the patient becoming
more self-aware or their condition in order to inhibit the external influences
potentiating their pain response (Ehde et al 2014). The patient learns a new skill
set to better disregard the outside reinforcement of the pain by learning tools for
relaxation, routine management, self-care, and the self-awareness of external
factors that potentiate their pain (Carlson 2008; Carlson et al 2001; Ehde et al
2014; Ullrich et al 2013).
10
Although somewhat effective, these therapies are still proving to be
unsatisfactory in treating all patients with continuous trigeminal neuropathic pain
(Koopman et al 2010). Therefore, a great need exists for new medications for
these patients and a greater understanding about this type of neuropathic pain
will better help to treat patients suffering with this type of continuous trigeminal
neuropathic pain. It is for such reasons that animal models of chronic pain
conditions such as trigeminal neuropathic pain need to be studied in more detail
to better understand the etiology and maintenance of these conditions as well as
to develop effective treatment strategies.
1.5.
Trigeminal Inflammatory Compression (TIC) Injury
It is necessary for pre-clinical models to accurately represent the clinical
picture of patients with trigeminal neuropathic pain in order to improve the validity
of the results obtained from pre-clinical research. The rodent model has been
chosen as the main model for inducing orofacial pain. One of the reasons for this
is due to the fact that rodents are readily available as well as having a similar
trigeminal system anatomy. Figure 1.5A depicts the rodent trigeminal system
demonstrating the three main branches that correspond to the three main
branches in humans (Leiser & Moxon 2006). Furthermore, it is quite easy to
study the histology of the trigeminal brainstem sensory nuclear complex in a
rodent because the barrel structure in the thalamus and brainstem corresponds
to the whisker barrels on the whisker pad ((Erzurumlu et al 2006; Lee et al
2009a; Lee et al 2009b; Li et al 1994; Negredo et al 2009; Zembrzycki et al
11
2013). Figure 1.5B (Mosconi et al 2010) illustrates the whisker barrels at the
trigeminal dorsal horn, thalamus, and cortex in relation to the whisker pad.
The historical model used to study trigeminal pain is the chronic
constriction injury of the infraorbital nerve (CCI-ION) (Donegan et al 2013;
Kernisant et al 2008; Vos et al 1994). This model induces mechanical allodynia
on the whisker pad of rats because chromic gut suture is used to loosely ligate
the infraorbital nerve (ION) as shown in Figure 1.6A (Kernisant et al 2008), a
nerve diverging off the maxillary branch. Although, this has been the most
popular trigeminal neuropathic pain injury model due to the robust induction of
chronic hypersensitivity, this model does have its weaknesses. First of all, this
injury does not induce hypersensitivity in all animals due to the difficulty of tying
the nerve. The discrepancy comes with the tightness of the ligation. For the rats
that do develop hypersensitivity, they do not develop allodynia until two to four
weeks post injury (Bennett & Xie 1988; Ma et al 2012b; Vos et al 1994). The first
two weeks the injured ION is silent, and it is the demyelination and other
peripheral and central neuroplastic and glial mediated events that cause the
hypersensitivity (Evans et al 2014; Taylor 2001; Uchida et al 2010a; Uchida et al
2010b). Furthermore, this procedure can only be performed in rats. Although, the
pCCI-ION model was adapted from the CCI-ION to be a specific surgery to be
performed in mice, this model punctures the ION and is difficult to repeat
because often times the partial ligation will cause a complete tear of the ION (Xu
et al 2008).
12
Our group, however, developed a new chronic trigeminal neuropathic pain
model for mice known as the Trigeminal Inflammatory Compression (TIC) injury.
The model is reliable, robust, and reproducible. Instead of ligating the nerve, as
in the CCI-ION, a small piece of chromic gut suture is placed in the fissure
between the maxillary bone and the infraorbital nerve. Figure 1.6B illustrates the
TIC injury with the chromic gut alongside the infraorbital nerve (Ma et al 2012a).
Our previous study determined the TIC injury model induces mechanical
allodynia in the mice within one week, which persisted through at least 10 weeks
in the initial study. Of the mice that receive this surgery, 100% of them develop
hypersensitivity. Lastly, we defined this not just as a neuropathic pain model, but
it also includes an inflammatory component because microglial activation is
observed in the trigeminal dorsal horn making this a very interesting model for
future continuing experiments (Ma et al 2012a).
1.6 .
Scope and Hypothesis of the Dissertation
The study described in this chapter of the dissertation will further
characterize the mouse TIC injury model as a suitable and effective model for
chronic, trigeminal neuropathic pain studies, and demonstrate its suitability for
evaluation of drugs that are efficacious in alleviating the mechanical allodynia in
the mice with TIC injury.
Behavioral characteristics of mice with the TIC injury were observed at
time points 1, 4, and 8 weeks post injury using cognitive dependent tests ((lightdark preference, open field, and elevated plus maze). Mice with TIC injury were
13
then given either FDA approved drugs (Pioglitazone and D-cycloserine) in
combination and/or separate to observe their inhibitory effects on orofacial
neuropathic pain.
Finally, assays were performed in isolated mitochondrial
preparations from the mice with TIC injury for comparisons to mitochondria from
naïve mice (treated vs. untreated) to determine the mitochondrial bioenergetics
of each group.
The hypotheses for this study are that:
1)
Orofacial
neuropathic
pain
after
TIC
injury
would
be
accompanied by anxiety- and depression-like behaviors.
2)
The activation of peroxisome proliferator-activated receptor
gamma (PPARγ) receptor would elicit anti-allodynic effects in the
mice with TIC injury. This was tested using the PPARγ agonist,
pioglitazone (PIO). The prediction is that: A. pioglitazone would
attenuate mechanical allodynia on the whisker pads of the mice
with TIC injury, B. PPARγ will be upregulated in the TBSNC, and
C. the combination of PIO and D-cycloserine (DCS), an Nmethyl-D-aspartate (NMDA) receptor agonist/antagonist, will
provide a potentiated analgesic effect.
3)
Mitochondrial dysfunction occurring in the mice with TIC is
partially responsible for the maintenance of chronic pain. This will
be tested using a mitochondrial uncoupler, 2,4-DNP to determine
its ability to attenuate the mechanical allodynia on the whisker
pads of the mice with TIC injury. Furthermore, this dysfunction
14
would be corrected with the PIO+DCS combination treatment ex
vivo utilizing the Seahorse XFe24 Analyzer.
15
Table 1.1. Chronic Pain is More Prevalent Than Other Major Diseases.
(Table posted by the AAMP).
Condition
Number of Sufferers
Source
Chronic Pain
100 million Americans
Institute of Medicine of The National
Academies
Diabetes
25.8 million Americans
American Diabetes Association
(diagnosed and estimated
undiagnosed)
Coronary Heart
16.3 million Americans
American Heart Association
Disease
(heart attack and chest
7.0 million Americans
pain)
Stroke
Cancer
11.9 million Americans
American Cancer Society
16
Figure 1.1. Anatomy of Trigeminal Nerve. The trigeminal system is composed
of three main nerve branches (V1-opthalamic, V2- maxillary, V3- mandibular) (a).
The facial dermatomes will synapse accordingly on the descending spinal nuclei
(b). All nerve meet at the trigeminal ganglion and synapse on the Trigeminal
Brainstem Sensory Nuclear Complex (TBSNC) composed of main sensory
nucleus, and descending spinal trigeminal nuclei (sp5): sp5 oralis, sp5
interpolaris, sp5 caudalis (c). (from de Leeuw, 2008, figure 1-1.)
17
Figure 1.2. Trigeminal Brainstem Sensory Nuclear Complex (TBSNC) with
Specific Nociceptor Fiber Representation. The majority of primary neurons
that synapse at trigeminal sp5 caudalis are C -fibers along with a few Aδ delta
fibers while the primary neurons that synapse at the main sensory nucleus, sp5
oralis, and sp5 interpolaris are primarily Aβ fibers. (from DaSilva and Dos Santos
2012, figure 1).
18
Figure 1.3. Functional Magnetic Resonance Imaging (fMRI) of Patient
Suffering from Trigeminal Neuropathic Pain. Activation areas (red) are
highlighted along the trigeminal pain pathway after a patient suffering with
orofacial pain receives a specific stimulus. The image shows that the
trigeminal ganglia (TG), trigeminal dorsal horn (spV), thalamus (Th), and
somatosensory (SI) cortex are activated after brush, cold, and heat stimuli
are applied to the face of patient suffering with orofacial pain. (from
Becerra, Morris et al. 2006, figure 2)
19
Figure 1.4. The Matrix of “Pain Sensation” and “Pain Affect.” The pain matrix
incorporates multiple brain regions integrating pain sensation and affect. The
areas shown are somatosensory cortex 1 and 2 (S1 and S2), posterior parietal
cortex (PPC), insula cortex, supplementary motor area (SMA), cingulate cortex
(ACC), prefrontal cortex (PFC), amygdala, thalamus, and periaqueductal gray
(PAG). (from May, 2009, figure 1).
20
A
B
Figure 1.5. The Trigeminal Sensory System in Rodents. (A) The rodent
trigeminal nerve separates into three branches similar to the human trigeminal
nerve (from Leiser and Moxon 2006, figure 1). (B) The barrel structure of whisker
pads of the rodents correspond to the barrel structures observed in the
brainstem, thalamus and cortex (from Mosconi, Woolsey et al. 2010, figure 1).
21
A
B
Figure 1.6. The CCI-ION Injury Model in Rats Compared to the TIC Injury
Model in Mice. (A) The CCI-ION model requires a loose ligation of the ION by
chromic gut suture that will induce hypersensitivity on the whisker pads of the
rats 2-4 weeks after surgery. (B) The TIC injury model places the chromic gut
suture alongside the ION to induce hypersensitivity on the whisker pads of the
mice within 1 week post-surgery. The arrows point to the ION and the chromic
gut suture. (figure A is from Kernisant, Gear et al. 2008, figure 1; figure B is from
Ma, Zhang et al. 2012, figure 1).
22
CHAPTER TWO
INDUCED FACIAL PAIN, ANXIETY- AND DEPRESSION RELATED
BEHAVIORS ASSOCIATED WITH TRIGEMINAL INFLAMMATORY
COMPRESSION (TIC) INJURY
2.1.
Introduction
Approximately 22% of the US population suffers from facial and
headache pain. Patients with trigeminal neuropathic pain, one type of chronic
facial pain, frequently report continuous aching and burning pain sensation that
may be accompanied by intermittent electrical shock-like experiences. Patients
who have this type of facial pain also suffer from mechanical allodynia and cold
hypersensitivity (Baron et al 2010; Zakrzewska 2013). While dental procedures
or trauma are known causes of peripheral trigeminal nerve injury and
inflammation, in some cases, no clear causes are identified for the origin and
maintenance of trigeminal neuropathic pain (Porto F 2011; Renton T 2011).
There are, however, a limited number of models of such pain conditions
available for use in laboratory experiments. Historically, one model of
neuropathic, facial pain frequently used in rats is known as the chronic
constriction injury of the infraorbital nerve (CCI-ION) (Vos et al 1994). This model
has been adapted for use in mice and is referred to as the partial CCI-ION (Xu et
al 2008). These models involve tying chromic gut suture around the ION, a
branch of the maxillary nerve which innervates the whisker pad of rats and mice,
causing mechanical hypersensitivity in the whisker pad region. However, the
23
suture causes deformation of the ION and constricts blood flow thus inducing
partial nerve ischemia and death (Bennett & Xie 1988; Kawamura 1997; Kim
1992), features not necessarily observed in patients suffering from trigeminal
neuropathic pain. To address these issues, a novel chronic facial neuropathic
pain model in mice, named as the Trigeminal Inflammatory Compression (TIC)
injury model, was developed in our laboratory to more closely mimic the clinical
characteristics of trigeminal neuropathic pain (Ma et al 2012a). As previously
reported, the TIC injury model is produced by inserting chromic gut suture
between the infraorbital nerve and the maxillary bone. This placement alongside
the nerve, rather than constriction of the nerve, has been successful in
preventing whole nerve ischemia, demyelination, and death in the small mice
(Ma et al 2012a).
Due to its novelty, there is a great need for the TIC injury model to be
further evaluated to increase our understanding of the behavioral characteristics
of the model. For example, one important aspect of the clinical presentation of
trigeminal neuropathic pain not yet evaluated is the common comorbidity of
psychological disorders and the emotional distress (Wall & Melzack 1999). In
clinical populations, symptoms of anxiety and depression in particular have been
consistently observed in patients with chronic trigeminal-mediated pain (Averill et
al 1996; Burris et al 2010; Fishbain 1999a; b; M. J. Robinson 2009; McWilliams
et al 2003; Nicholson & Verma 2004).
The measurement of constructs such as anxiety and depression in animal
models, however, has proven more difficult than making these measurements in
24
clinical populations. Fortunately, the use of cognitive dependent tests offers a
more thorough examination of psychological constructs such a depression and
anxiety, and are increasingly used by researchers seeking to understand chronic
neuropathic pain conditions (Mao et al 2008; Mogil 2009). Anxiety-like behaviors
in animals have been extensively studied, and numerous ones resulted in
validate protocols (Belzung & Griebel 2001). Three assays that are particularly
well understood in measuring animal behavior associated with psychological
constructs are: the light-dark box preference test, the open field exploratory test,
and the elevated plus maze task. Furthermore, the activity and rearing behavior
in each of these tasks has been previously shown to be affected by the
experience of pain (Bouwknecht & Paylor 2002; Crawley 1980; Parent et al 2012;
Roeska et al 2008).
The aim of the current study was to further characterize the novel TIC
injury model by examining mechanical allodynia and thermal hypersensitivity and
by measuring pain-related anxiety- and depressive-like behavior with cognitive
dependent tests. My hypothesis was that mice with TIC injury would display
greater mechanical allodynia, cold hypersensitivity, and more anxiety- and
depressive-like behaviors than that of naïve mice or animals undergoing sham
surgical procedures only.
25
2.2. Materials and Methods
2.2.1. Animals
All experiments were performed with C57Bl/6 male, wild-type mice that
weighed between 25 and 35 grams purchased from Harlan Laboratories
(Indianapolis,
IN). Animals
were
randomly assigned
to receive
either
experimental (TIC injury model) surgical procedures, sham surgical procedures,
or to remain in the naïve cohort. Mice were housed in a well-ventilated mouse
housing room (maintained at 27oC) with a reversed 10/14 h dark/light cycle so
that testing could be performed in their active period. All mice had access to food
and water ad libitum throughout the duration of the experiment. Low soy bean
content diet normal chow was provided (Teklab 8626, Harlan, Indiana). All
experimental procedures were completed according to the guidelines provided by
the National Institute of Health (NIH) regarding the care and use of animals for
experimental procedures. Animal protocols were approved by the University of
Kentucky’s Institutional Animal Care and Use Committee (IACUC). All animals
were housed in facilities approved by the Association for Assessment and
Accreditation of Laboratory Animal Care International (AAALAC) and the United
States Department of Agriculture (USDA).
2.2.2. Trigeminal Inflammatory Compression (TIC) Injury Surgery
Mice were anesthetized with sodium pentobarbital (70 mg/kg, i.p.). Under
the standard sterilized condition, the hair on the top of their head was then
26
shaved, and ophthalmic cream was applied over their eyes to protect the eye
from over-dry. Mice were then fully constrained in a stereotaxic frame. A small 15
mm incision was made along the midline of the head and the orbicularis occuli
muscle was gently dissected and retracted away from the bone. Small cotton
balls were packed into the orbital cavity to control bleeding, and the infraorbital
nerve was located approximately 5 mm deep against the bony fissure. Animals
randomly assigned to receive the TIC injury surgery underwent surgical
placement of a 2 mm length of chromic gut suture (6-0), inserted between the
infraorbital nerve and the maxillary bone infraorbital fissure. Chromic gut suture
was inserted specifically in this region to adhere to specific infraorbital nerve
bundles in order to prevent the chromic gut suture from being lost in the orbital
cavity, but not to pierce the entire infraorbital nerve. Mechanical allodynia was
induced in the mouse whisker pad due to the suture physically stimulating the
nerve as well as the chromate salt released from the suture. Animals assigned to
receive sham surgical procedures did not have the chromic gut suture
placement, but only received the skin incision and muscle dissection. Naive
animals did not receive any surgery. All mice were aged matched.
2.2.3. Behavioral Tests
All behavioral tests were conducted during the animal’s active cycle (i.e.
dark phase of the dark/light cycle) during the hours of 8:00 am to 6:00 pm. During
testing, either a red-light or a dim lamp was illuminated to allow light for the
experimenters. None of the behavioral tests were conducted on the same day.
27
2.2.3.1. Assessment of Mechanical Allodynia
Mechanical threshold of the whisker pad was measured before and after
surgery with a modified up/down method (Chaplan et al 1994) using a graded
series of von Frey fiber filaments (force:0.008 g (size:1.65); 0.02 g (2.36); 0.07 g
(2.83); 0.16 g (3.22); 0.4 g (3.61); 1.0 g (4.08); 2.0 g (4.31); 6.0 g (4.74);
Stoelting, Wood Dale, IL). One experimenter gently restrained the mouse in their
palm (2-5 minutes) with a cotton glove until the mouse was acclimated and calm.
A second experimenter applied the von Frey filaments to the mouse’s whisker
pad. The 0.16 g (3.22) fiber was applied first. If the mouse responded three or
more times out of five trials to the fiber, this was considered to be a positive
response and the next lower gram force filament was applied. However, if the
mouse responded two or fewer times out of five to the fiber applied, this was
recorded as a negative response and then the filament with the next higher gram
force was applied. Head withdrawal/front paw sweeping/biting all were
considered positive responses. Time between applications of each filament was
2-10 seconds. After one fiber successfully caused positive responses, application
of the subsequent fibers continued until four fibers were applied or until the
animal responded to the lowest gram force fiber. Data were analyzed with a
curve-fitting algorithm that allowed for estimation of the 50% mechanical
withdrawal threshold (measured in gram force). The decreased mechanical
threshold value is an index of mechanical allodynia. Responses to the von Frey
fibers stimulations were recorded on day 7 post surgery (TIC and sham) and
continued once a week post-surgery testing both the ipsilateral and contralateral
28
whisker pads. A cohort of naïve mice were tested intermittently (weeks 2, 4, 8,
10, 11) alongside the sham animals and mice with TIC.
2.2.3.2. Assessment of Thermal Hypersensitivity
The protocol used to measure thermal hypersensitivity was adapted from
Neubert and colleagues (2005) (Neubert et al 2005). Neubert and colleagues
used a metal probe connected to a water bath placed in an operant licking box.
The present study utilized a looped copper coil probe (0.065” I.D.; 1/8” O.D.;
0.030” Wall Thickness, Restek Corporation, Bellefonte, PA) with a 5 x 3 x 3 mm
tip connected to an insulated rubber tubing and attached directly to an Isotemp
bath circulator (39.5 x 24.5 x 39 cm, Isotemp 3016S; Fisher Scientific). The water
bath was filled with antifreeze liquid, and digitally set to a specified temperature
for testing. The temperature of the copper-wire probe was measured with a
Physiotemp temperature monitor (Thermalert Model TH-8; PHYSITEMP
INSTRUMENTS INC. Clifton, NJ, USA). The temperatures reported in this study
were measured from the tip of the copper-wire probe. The 10-11 ᵒC was chosen
to activate cold nociceptors; likewise 45-46.5 ᵒC was chosen to activate heat
nociceptors (Neubert et al 2005; Rossi & Neubert 2009). Room temperature (2324.3 ᵒC) and body skin temperature (32-32.5ᵒC) were chosen to act as controls
for this experiment to ensure that the probe was not causing an adverse
mechanical response to the animals. The mouse skin was shaved on the
ipsilateral V2 (trigeminal nerve second branch) region just behind the whisker
pad 24 hours before testing (Cha et al 2012). One experimenter gently restrained
29
the mouse in their palm with a cotton glove until the mouse was acclimated and
calm (5 minutes). The other experimenter applied the looped copper-wire probe
to the shaved V2 region (Figure 2.1.A). Head withdrawal latency, the time in
seconds from which the stimulus was applied to the time the mouse reacted with
head withdrawal/front paw sweeping/biting, was recorded. Three trials for each
temperature were conducted with 1 minute intervals between each trial. Only one
temperature was recorded per testing day and all thermal testing occurred after
post-operative week 8.
2.2.3.3. Acoustic Startle Disturbance
The acoustic startle disturbance test is a well-established measure of
anxiety-like behaviors in response to a stressful stimulus (Blaszczyk et al 2010;
Blaszczyk et al 2000; Geyer et al 1982). Mice were placed in a vinyl cylinder
container (radius: 21.5 cm; depth: 29.9 cm) with room for the animal to move.
Using a modified form of an acoustic startle disturbance, one experimenter
pressed a © Top Paw Dog Training Clicker (2" Length “blue bone” clicker; Item #
39330 purchased from ©Pet Smart, Lexington, KY) above the animal irregularly
for a period of 2 min. The clicker was pressed with force eliciting an average
frequency of 430.89 Hz (recorded with a KAYPENTAX COMPUTERIZED
SPEECH LAB, Real-Time Pitch; Model #5121, version 3.4.1) ranging from 70110 dB (Decibel Meter App; Version 1.6; Device Type: iPhone4 iOS Version:
6.1.3). Mice not currently being tested were housed in a separate sound-proof
room during the acoustic startle test. Immediately after exposure to the mild
30
acoustic startle disturbance, mice were either placed in the dark side of the lightdark box or in the central area of the elevated plus maze to begin the test
designated for that day. Only one of the operant tests was conducted per day.
2.2.3.4. Two Compartment Light-Dark Box Preference Test
The light-dark box preference task has a long history of use in the
measurement of anxiety-like behavior. In this task, anxiety-like behaviors are
believed to manifest as a decrease in: 1) total time spent in the light area, 2)
number of entries into the light area, and 3) number of rearing/exploratory
behavior (Bouwknecht & Paylor 2002; Crawley 1980; Hascoet 1998). The lightdark box consisted of two equally-sized chambers (one illuminated and one
darkened; 11 x 19 x 12 cm/each) connected with a 5 x 5 cm doorway in which
mice were allowed to freely move between chambers. Immediately after
exposure to the mild acoustic startle disturbance (described above), mice were
placed in the dark side of the box facing away from the light chamber. Animals
remained in the light-dark preference box for a total of 10 minutes. Behaviors
measured in this test included: (1) time spent in the light area, (2) number of
transitions into the light and dark chambers, defined as at least partial passage
between chambers with extension of at least one of the animal’s back leg from
one chamber to the next; (3) number of rearing events, a measurement for
exploratory behavior, (4) latency of the first transition into the light chamber, and
(5) latency of first re-entry (transition) back into the dark chamber. Behaviors
were measured at post-operative weeks 1, 4, and 8.
31
2.2.3.5. Open Field Exploratory Activities
Exploratory behaviors were measured using a Flexfield Animal Activity
System (San Diego Instruments, San Diego, CA). This apparatus consists of
two Plexiglas chambers (40 x 40 x 36 cm) equipped with Photobeam Activity
System (PAS) software coupled to a Compaq 486 computer (Hewlette Packard,
Palo Alto, CA). Each chamber contained infrared photobeam sensors with 16
beams on each axis (total of 32 beams) that are arranged 1.25 cm above the
chamber floor. Obstruction of these photo beams constitutes movements in the
x- and y- plane. The x- and y- plane were divided into 5 zones to define the
center and peripheral area. Another set of 16 beams is located 8 cm above the
chamber floor to record movements along the z-axis measured i.e. rearing
events and rearing duration (Zhang et al 2004). Data were collected in 5-min
intervals for a total of 45 minutes to record: (1) number and duration of rearing
(2) active time vs. rest time, (3) overall distance travelled, (4) total beam breaks,
and (5) time spent in the central verses peripheral areas of the chamber. The
sum of time spent in Zone 1-4 equaled the total duration of time spent in the
periphery. The duration of time in Zone 5 equaled the duration of time spent in
the center. Behaviors were measured at post-operative weeks 1, 4, and 8.
32
2.2.3.6. Elevated Plus Maze Task
The elevated plus maze task is a widely used test for measuring fear and
anxiety-like behavior and has been previously shown to be affected by rodent
pain models (Belzung & Griebel 2001; Kontinen et al 1999; Parent et al 2012;
Roeska et al 2008; Walf & Frye 2007). The elevated plus maze (Bioseb, Vitrolles,
France) consists of four arm cross-shaped device (length: 35 cm, width: 5
cm/each, height from floor: 51 cm); two arms are enclosed on three sides by 15
cm high walls and the other two are not. All arms meet in a central area (5 cm x 5
cm) which allows animals to move freely throughout each zone of the maze. A
computer equipped with automated program software (BIOEPM 1.1.14; BIOSEB,
France) and linked with a camera head (DFK22AUC03) recorded each animal’s
movement throughout the maze. In this test, the open arms represent a
potentially threatening environment and thus anxiety-like behavior in response to
this threat is believed to manifest as a decrease in the time spent in and the
number of entries into the open arms of the maze (Belzung & Griebel 2001).
Immediately after the acoustic startle disturbance (described above), mice were
placed in the central area of the maze and allowed to explore the maze for a
period of 5 minutes. Mouse behaviors were coded and analyzed off line by a
blinded observer for: (1) time spent in open arms, (2) number of transitions into
open and closed arms, (3) number of head dips into the open arms, defined as
the movement of the animals head from the closed arm to the open arm of the
maze. Behaviors were measured at post-operative weeks 1, 4, and 8.
33
2.2.4. Statistical Analysis
The GraphPad Prism 6 statistical program was used for all data analysis
(Graph Pad Software, Inc. La Jolla, CA). Results are shown as the mean ±
standard error of the mean (S.E.M.). Data were analyzed by a one-way analysis
of variance (ANOVA) tests followed by Tukey post hoc test and two-way ANOVA
followed by Fishers post hoc test (where was appropriate). A p<0.05 was
considered significant for all tests.
2.3.
Results
2.3.1. Animals with TIC Injury Displayed Unilateral Whisker Pads Mechanical
Allodynia
All mice (100%) that underwent TIC surgery developed mechanical
allodynia on of the ipsilateral but not contralateral whisker pad as determined
with von Frey fibers thus confirming the results of our previously published paper
for this model (Ma et al 2012a). For all mice with TIC injury, the mean 50%
mechanical threshold of the ipsilateral side was 0.03 ± 0.28g an indication of
mechanical allodynia; while the mean 50% mechanical threshold of the
contralateral side was 3.72 ± 0.12g (n=13) and did not change from baseline
(Figure 2.1B). The mechanical threshold of the ipsilateral side of the mice with
TIC injury were significantly different from the sham animals (ipsilateral, 3.36 ±
0.07g; contralateral, 3.49 ± 0.05g; n=12) and naïve animals (ipsilateral, 3.55 ±
0.019g; contralateral, 3.50 ± 0.21g, n=5; p<0.0001, two way ANOVA, Fishers
34
post hoc test) within one week post injury as previously reported (Ma et al
2012a). The chronicity of this model was further shown by decreased mechanical
threshold in animals with TIC injury that was present post injury only on the
ipsilateral side (until the animals are euthanized at week 21).
The previous study published (Ma et al 2012a) mentioned the presence of
a receptive field for each mouse, but was not discussed in detail. It is important to
note that all mice with TIC responded to fiber 0.4 g (3.61) regardless of specific
receptive field stimulation. In order to find the specific receptive fields of an
injured animal, the von Frey fibers <0.16 g (3.22) were applied to the whisker pad
sporadically until a withdrawal response occurred. The sham and naïve animals
did not respond to fiber 0.4 g (3.61). The location of the receptive field for each
animal was always the same every week and rarely changed once established
confirming that the location of the chromic gut suture is not only injuring a
localized area of axonal fibers along the infraorbital nerve, but also that these
fibers correlate with a specific receptive field on the whisker pad.
Three different patterns of sensitivity were identified on the receptive fields
of the whisker pads of the injured mice (Figure 2.1C). Mouse #1 is
representative of the most typical receptive field observed (50% of the mice with
TIC injury had this phenotype). The second most common pattern is represented
by Mouse #2 (38% of mice with TIC). However, Mouse #3 (11% of mice with TIC)
represents a uniquely different phenotype than the previous two in that its
receptive fields are spread sporadically throughout the whisker pad. Interestingly,
all mice (100%) subjected to TIC surgery showed a sensitive spot at the 6 o’clock
35
position on the whisker pad (Figure 2.1C, indicated by the red x with a circle).
The different pattern of receptive fields did not change over the course of the
animal’s life.
2.3.2. TIC Injury Induced Unilateral Cold Allodynia of the V2 Area
Withdrawal latencies for all thermal stimuli were recorded (n=8; Figure
1D). In the 10-11ᵒC temperature stimuli point, the head withdrawal latency of
animals with TIC injury (5.11 ± 0.62s) was significantly different compared to that
of sham animals (9.21 ± 0.88s) and naïve animals (9.75 ± 0.71s; p<0.001, one
way ANOVA, Tukey post hoc test). The head withdrawal latency of animals with
TIC for 45-46.5 ᵒC temperature point was 10.21± 1.20s which was not significant
compared to that of sham animals (9.42 ±1.11s) and naïve animals (8.63 ±0.90s;
p>0.05). At room temperature stimuli point (23.24.3ᵒC), the head withdrawal
latency of animals with TIC injury (15.54 ± 1.52s) were similar to that of sham
animals (79 ± 0.83s) and naïve animals (17.17 ± 1.13s; p>0.05). At mouse skin
temperature (32-32.5ᵒC) point, the head withdrawal latency of animals with TIC
injury was 20.42 ± 2.37s. This was not significantly different from that of the
sham (17.25 ± 1.87s) and naïve animals (15.92 ± 1.75s; p>0.05; Figure 2.1D).
The results indicated that animals with TIC injury were sensitive to mild cold
stimuli, but had normal reactions to heat, room temperature, and skin
temperature.
36
2.3.3. Animals with TIC Injury Displayed Anxiety- and Depressive-Like Behaviors
in Two Compartment Light-Dark Preference Testing
Immediately after the 2-min acoustic disturbance the mice were put in the
dark side of the light-dark box facing away from the light box and the 10 min
experiment began. Total time spent in light and dark sides, light-dark transitions,
rearing events and latencies of first cross into light and re-entry into dark were
measured over the 10 min. There were no significant differences in time spent in
the light or numbers of rearing events in weeks 1 or 4 post injury. There was no
significant difference between mice with TIC and sham animals when comparing
latency of the first cross into the light, latency to re-enter dark side, or the number
of light-dark transitions (Table 2.1). Both mice with TIC and sham animals spent
almost 50/50 time in the light or dark side of the box at post-operative week 1
and week 4. Until post injury week 8, mice with TIC injury spent less time in the
light, 231.60 ± 25.55s, i.e. they hide in the dark chamber (over 70% of testing
time). In contrast, the sham mice spent as long as 330.39 ± 43.72s (over 50%) in
the light chamber (TIC vs. sham P<0.05, Two-way ANOVA, Fishers post hoc
test, Figure 2.2A). Mice with TIC injury also had significantly less rearing events
(9.77 ± 1.89/10min) at week 8 post injury compared to sham animals at week 8
post operation (18.82 ±1.95/10min, Figure 2.2B). When the number of rearing
events were analyzed by the first and second five minute intervals of the lightdark box preference, mice with TIC injury still showed significantly fewer numbers
of rearing events compared to sham animals (Table 2.1). The results of the light-
37
dark box preference test indicated that mice with TIC injury develop signs
associated with anxiety- and depressive-like behaviors 8 weeks after injury.
2.3.4. Acoustic Disturbance Affected Mice with TIC in the Elevated Plus Maze
Task
Immediately after the 2 min acoustic disturbance, both mice with TIC injury
and sham groups were subjected to an acoustic disturbance on post-operative
weeks 1, 4, and 8. Mice with TIC injury spent a significant time in the open arm
(78.90 ± 30.45s) compared to that of sham animals (8.89 ± 3.52s, p<0.01, Twoway ANOVA, Fishers post hoc test) at week 1 post operation (Figure 2.3A).
Mice with TIC injury also had a greater number of transitions into the open arms
at week 1 (4.00 ± 0.67/5min) and week 4 (2.09 ± 0.69/5min) compared to sham
animals (week 1, 1.50 ± 0.42/5min; week 4, 0.25 ± 0.16/5min; p<0.001, Two-way
ANOVA, Fishers; Figure 2.3B). The number of head dips into the open arm did
not differ between the mice with TIC injury and the sham animals at weeks 1, 4,
an 8 post injury (Figure 2.3C). However, in a separate cohort of animals that did
not receive the acoustic disturbance stimulation before the elevated plus maze
task, a significant difference was not observed in time spent in the open arms
and the number of transitions when comparing mice with TIC injury and sham
animals (Table 2.2). These results suggest that the mice with TIC injury
experience an extinction of fear behavior only after an acoustic disturbance at
week 1 and 4 post injury.
38
2.3.5. Mice with TIC Injury Showed Less Exploratory Activities
Animals with TIC injury also showed a decrease in rearing duration
(153.11 ± 18.22s) and number of rearing events (229.89 ± 27.36) at week 8
compared to the sham animals (rearing duration, 224.75 ± 25.09s; rearing
events, 337.46 ± 37.67; P<0.05; Figure 2.4A & 4B). Mice with TIC injury had
decreased active time (2311.50 ± 63.94s) and increased resting time (388.48 ±
63.94) 8 weeks after injury compared to that of sham animals (active, 2513.30 ±
43.15s; resting, 186.72 ± 43.15s; Figure 2.4C & 4D). Mice with TIC injury also
had decreased total distance traveled compared to sham animals (TIC: 1370.20
± 110.60cm; sham: 1666.00 ± 114.38cm, P<0.05; P<0.01; Two-way ANOVA,
Fishers post hoc test; Figure2.4E). A significant difference at week 1 or 4 post
injury was not observed (p’s>0.5). There was also no difference in total beam
breaks or center vs. peripheral time duration at any time point (Figure 2.4F, 4G,
& 4H). The open field results parallel the light-dark box data supporting the
development of depressive-like behaviors in mice with TIC injury at week 8 post
injury.
2.4. Discussion
2.4.1 TIC Injury Model Mimics Clinical Facial Neuropathic Pain
In the present study, we determined that the surgery is 100% efficacious
for inducing hypersensitivity in all of the animals receiving the chromic gut suture
placement. All mice experienced mechanical allodynia with distinctive receptive
39
fields (sensitivity spots) that did not change over the course of the animal’s life.
However, the receptive field pattern variations in the TIC model were due to the
relative position of the chromic gut suture, the maxillary bone, and the infraorbital
nerve. The development of cold allodynia in the mice with TIC injury, but not heat
hypersensitivity was another important feature of this model. Clinic patients with
neuropathic pain experience cold hypersensitivity but not heat sensitivity (Baron
et al 2010; Zakrzewska 2013). We also initially determined that the chronicity of
the TIC injury model lasts at least until 21 weeks. These were the first recorded
data indicating a mouse facial pain persisting with this time course without
causing ischemia and complete demyelination of the infraorbital nerve (Ma et al
2012a).
Many acute and chronic orofacial models have existed that successful
induce hypersensitivity in the mouse. However, many of the acute models do not
last longer than a week and fall short of reflecting the full clinical characteristics
of chronic facial pain (Bornhof et al 2011; Luccarini et al 2006; Quintans et al
2014). The chronic models of facial pain have also either differed significantly
from the clinical picture of patients with trigeminal pain or have had low efficacy
in mice (Saito et al 2008; Siqueira-Lima et al 2014; Xu et al 2008; Zhang et al
2012b). The TIC injury model is beneficial because it is not only reliable and
reproducible, but it also closely mimics the clinical presentation of this pain
condition. Thus, this study provides support improving the validity for using the
model.
40
The TIC model demonstrated several aspects consistent with chronic
facial pain in humans. While the mice with TIC had distinct receptive fields to
elicit a withdrawal reflex response, human patients also have specific receptive
fields triggering pain. Likewise, the pattern of the receptive fields were not always
represented the same in all animals, as the pattern of receptive fields from
person to person may also vary (Simons & Travell 1981; Siqueira et al 2009;
Travell 1981). However, the receptive fields in the mice with TIC did not vary over
the course of the mouse’s life providing a reliable sensitivity area for testing
evoked hypersensitivity over a chronic period of time. Moreover, as the mice with
TIC suffered from only cold hypersensitivity, patients suffering with trigeminal
neuropathic pain are not often bothered by heat stimuli, but instead most often
complain that light touch, wind, or cold air trigger a shooting pain (De Leeuw
2008; Zakrzewska 2013).
2.4.2. TIC Injury Developed Anxiety- and Depressive -Like Behaviors Similar to
Patients Suffering with Chronic Facial Pain
For the light-dark box preference and open field exploratory test, the
behavior of the sham mice in weeks 1, 4, and 8 post operation remained
relatively consistent, but was significantly altered in the mice with TIC injury at
week 8 post injury. In the light-dark box preference test, the mice with TIC injury
spent significantly less time in the light side of the box and had a fewer number of
rearing events. Similarly for the open field testing, the mice with TIC showed a
decrease in number of rearing events, decreased active time with increased
41
resting time, and a decreased total distance traveled only at post injury week 8.
Although, cognitive dependent behavioral testing beyond week 8 is not reported
in this paper, these anxiety-and depressive-like behaviors remained consistent
throughout the remainder of the animals’ lives parallel with the whisker pad
sensitization that occurs after the TIC injury.
Previous research has shown that a decreased amount of time spent in
the light side of the light-dark box is indicative of anxiety-like behavior while
decreased number of rearing events and transitions into the light side are
indicative of depressive-like behavior (Costall & Naylor 1997; Cryan & Holmes
2005; Fedorova et al 2003). Open field has been used to measure the
exploratory and locomotor activity of animals including number of rearing events,
active vs. resting time, and center vs. peripheral time. Decreased number of
rearing events, decreased active time, and decreased occupancy center time is
considered a reliable index of anxiety-like behavior and a measure of response to
anxiolytic agents (Costall & Naylor 1997; Katz & Roth 1979; Ramos et al 1997).
Together, the results of the light-dark box and open field testing have supported
that the chronicity of the TIC injury producing anxiety- and depressive-like
behaviors starting at week 8 post injury. Other chronic pain animal models have
also observed similar depressive-like behavior after migraine-induced pain,
sciatic nerve injury, etc.(Dellarole et al 2014; Lipton et al 2000; McWilliams et al
2003; McWilliams et al 2004; Robinson et al 1988). Furthermore, Yalcin and
colleagues (2011) similarly reported a time course of chronic pain animals
revealing the development of depressive-like behaviors also occurring 6-8 weeks
42
post injury (Yalcin et al 2011). These data compared to other studies reported
suggest that it is the chronicity of the TIC injury model that allows the
development of the “pain affect” to ensue as another facet observed with
similarity to patients with chronic pain.
Wall & Melzack (1999) described chronic pain as having two main
features: “pain sensation” and the “pain affect” which has incorporated the
emotional distress the patients undergo due to the pain (Wall & Melzack 1999).
The “pain affect” has also been described as the sequela of other physical and
psychological disorders such as anxiety and depression (Dellarole et al 2014;
Maletic & Raison 2009; McWilliams et al 2003; McWilliams et al 2004). These
comorbidities have been so prevalent that approximately half of all patients
suffering from chronic pain have also suffered from anxiety and depression
((Asmundson & Katz 2009; Asmundson & Taylor 2009; Macianskyte et al 2011;
Robinson et al 2009).
The relationship between chronic pain, depression,
anxiety, and fear is extremely complex with extensive overlap making it difficult to
determine a pattern of cause and effect. This complex relationship could be
related to the numerous brain structures, such as the somatosensory cortex,
prefrontal cortex, nucleus accumbens, insular cortex, anterior cingulate cortex,
amygdala, hippocampus, thalamus, and cerebellum involved in pain perception
have all also been highlighted as major players in affective states, including
depression, anxiety, and fear (Apkarian 2004; Apkarian et al 2004a; Apkarian et
al 2004b; Averill et al 1996; Becerra et al 2006; Dellarole et al 2014; Fishbain
1999a; b; Robinson et al 2009). Thus, the TIC injury model seems to be a
43
representative model of anxiety-related chronic facial pain. This makes it a good
model for testing pharmaceutical agents with potential for facial pain relief, and
also for testing anxiolytics agents, such selective serotonin reuptake inhibitors
(SSRIs), and other antidepressants to attenuate pain as well as comorbid
psychological effects.
2.4.3. Decreased Fear Response in Mice with TIC after Acoustic Disturbance
The development of the anxiety- and depressive-like behaviors at week 8
post injury was interesting considering that the elevated plus maze results
showed opposite behavior at weeks 1 and 4 post injury. Mice with TIC injury
spent more time in the open arm at week 1 post injury and had a significantly
greater number of transitions at weeks 1 and 4 post injury compared with that of
sham mice. One could argue that these results represented anxiety-like behavior
in our sham animals (Table 2.2); likewise, the sham animals were not
significantly different from the naïve animals in time spent in open arm and
number of transitions. Therefore, the data strongly support the idea that the TIC
injury was the cause of the behaviors observed. Interestingly, our results in the
elevated plus maze suggest that after acoustic disturbance, the mice with TIC
injury were showing evidence for decreased anxiety at week 1 and possibly week
4 post injury (Bailey & Crawley 2009; Davis et al 1997). Mice with TIC that were
not exposed to the acoustic disturbance, however, did not show a significant
difference in time spent in the open arm or number of transitions compared with
that of sham animals also not exposed. These results raise some interesting
44
questions: 1) Why was there decreased anxiety-like behaviors at week 1 post
injury and increased anxiety-like behaviors at week 8 post injury in the mice with
TIC only after an acoustic disturbance? 2) Were the behaviors in the elevated
plus maze task not anxiety-like behaviors at all? I offer possible reasons for these
results in the discussion below.
One explanation is that the mice with TIC were exposed to two additional
stressful stimuli in an already sensitized state. The first stressful stimulus was the
mild acoustic startle stimulation. An acoustic startle disturbance has been shown
to create a “stress-induced analgesia” by inducing endogenous opioids release,
thereby, creating the possibility that the animal is in a less painful condition (Frew
& Drummond 2008; Lewis et al 1980; Nencini et al 1984; Terman et al 1984;
Vitale et al 2005; Watkins & Mayer 1982). The acoustic stimulus has also been
shown to activate the HPA axis which would not only cause an increase in
activity, but also would similarly decrease neuropathic pain (Buijs et al 1993;
Buijs & Van Eden 2000; Gonzales et al 2008; Kosten & Ambrosio 2002). Since
the separate cohort of animals who did not receive the acoustic disturbance did
not have the same behavioral results, the acoustic disturbance might have
induced a temporary “stressed-induced analgesia” as a potential reason for the
increased time spent in open arms and increased number of transitions.
Although, the acoustic stimulation did not appear to have much effect on
the mice behaviors in the light-dark box preference test, it was possibly an
important stressor in the elevated plus maze test, the second stressful stimulus.
This test was more stressful than the light-dark box and open field, and the
45
former could have triggered anxiety-like activities that the latter two did not (Cruz
et al 1994; Rodgers 1997; Rodgers & Dalvi 1997; Salas et al 2003; Walf & Frye
2007). Thus, this would contribute to the results observed in the elevated plus
maze at weeks 1 and 4 after injury in the mice with TIC. The increased number of
transitions into the open arm possibly suggests hyperactivity, and the increased
time spent in open arm could suggest a temporary decrease in pain sensation.
Another possible explanation for these data was that there was a
decrease in fear/defensive behavior due to poor decision making in the injured
animals after the acoustic startle stimulation in the elevated plus maze task. This
could be due to lack of fear associated with increased release of endogenous
opioids or due to major sites in the brain which might be undergoing plasticity at
different time points following the injury. Neugebauer and colleagues (2004)
described the amygdala as being a multi-functional integration site (Neugebauer
et al 2004). The pre-frontal cortex, amygdala, and ventral hippocampus are just a
few areas of the brain crucial in fear and stress responsive behaviors (Ji et al
2010; Neugebauer et al 2004; Quirk et al 2000; Rea et al 2013). Rodents that
have received excitotoxic lesions localized to the pre-frontal cortex, amygdala,
and ventral hippocampus have also shown a significant more time spent in open
arms and increased number transition compared with that of sham animals
(Quinn et al 2002; Shah & Treit 2003; Ventura-Silva et al 2013).
Negative
emotions such as anxiety and depression facilitated pain, but the negative
emotions associate with fear and stress promoted the inhibitory pathways of the
amygdala which then inhibited pain. This interpretation allows for a possible
46
coexistence of depression, fear, and pain. Therefore, if certain pharmaceutical
agents were devised or used that not only treated depression, but also pain, then
perhaps our understanding of these mechanisms and treatment would be
improved.
2.5. Conclusion
In summary, due to the efficacy and persistence that is translationally
relevant to the chronicity of the clinical injury, the TIC injury model is a good
chronic trigeminal pain model that can be used to determine different signaling
cascades initiated during the course of facial pain. This will help not only to
identify molecular targets, but also to define differences between facial pains
versus somatic pain relayed by the spinal cord after nerve injury. Since the TIC
injury model has been developed in mice, gene therapies can be used in order to
target particular cytokines, proteins, and receptors in the hopes of revealing
underlying mechanisms and how specific gene knock-outs influence trigeminal
pain. Furthermore, since this model displays anxiety- and depressive-like
behaviors by 8 weeks, it is a useful model to study certain pharmaceutical agents
that perhaps can help attenuate not only depression/anxiety, but also fear,
anxiety, and depression related to pain.
47
Table 2.1. Light-Dark Box Data.
Week
Group
Latency to
first cross
(s)
Light/dark
Transitions
(#)
Latency to
re-enter
dark side
(s)
Rearing Events
1
st
nd
2
0
Naïve
41.53±12.45
9.88±1.37
21.06±8.70
11.88±1.61
6.00±1.12
1
TIC
56.17±12.53
10.38±1.29
21.09±3.77
9.33±0.81
7.57±1.08
1
SHAM
40.55±13.91
11.13±1.29
20.29±5.67
9.88±1.42
8.00±1.00
4
TIC
23.48±9.88
9.83±1.90
65.66±24.86
10.58±1.54
6.5±1.91
4
SHAM
23.85±10.26
11.17±1.36
48.64±35.44
10.42±1.36
6.50±1.12
8
TIC
35.52±11.70
9.38±1.47
31.61±16.18
5.46±1.23
4.31±0.77
8
SHAM
13.08±5.47
11.55±1.89
18.33±3.59
9.73±1.24
9.09±0.97
48
Table 2. 2. Elevated Plus Maze Data.
Week
Group
Open Arm
Duration (s)
Transitions
into Open
Arms (#)
Head Dips into
Open Arms (#)
0
Naïve
21.43±7.86
2.38±0.75
10.75±0.99
1
TIC
78.90±30.45
4.00±0.67
8.36±1.52
1
SHAM
8.89±3.52
1.5±0.42
11.38±1.60
4
TIC
17.11±5.58
2.09±0.69
6.18±1.03
4
SHAM
0.93±0.61
0.25±0.16
4.13±0.90
4
TIC
(NS)
8.13±4.66
0.63±0.32
6.00±1.82
4
SHAM
(NS)
0.00±0.00
0.00±0.00
5.50±0.98
8
TIC
1.44±0.79
0.45±0.21
5.64±1.13
8
SHAM
0.00±0.00
0.00±0.00
5.00±1.05
8
TIC
(NS)
0.887±0.89
0.13±0.13
3.13±0.44
8
SHAM
(NS)
1.94±1.31
0.25±0.16
2.38±0.38
49
B
C
D
12
3
9
6
#1
50%
H e a d W ith d r a w a l L a t e n c y ( s )
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
A
6
N a iv e I p s ila t e r a l
N a iv e C o n t r a la t e r a l
S H A M I p s ila t e r a l
S H A M C o n tr a la t e r a l
T I C I p s ila te r a l
T I C C o n t r a la te r a l
5
4
3
2
****
1
0
0 1 2 3 4 5 6 7 8 9 1011
T im e ( w e e k s )
21
25
N A IV E
SH AM
20
T IC
15
10
5
***
0
1 0 -1 1
2 3 -2 4 . 3
3 2 -3 2 . 5
4 5 -4 6 . 5
T e m p e r a tu r e ( ° C )
#2
38%
#3
11%
Figure 2.1. TIC Injury Induced Mechanical and Cold Allodynia. The mice with
TIC were tested on the whisker pad for mechanical allodynia and thermal
hypersensitivity in the V2 area behind the whisker pad (A). The ipsilateral
whisker pad of the mice with TIC had a significant decrease in mechanical
threshold than that of their contralateral side and the sham and naïve mice
50
starting within the first week post injury lasting until euthanasia, 21weeks post
injury (B). The patterns variations of the receptive fields are shown in (C) with
100% of the mice receiving the TIC surgery developing allodynia. Mice with TIC
are only hypersensitive to cold temperatures (D). n= 7-10; Two way ANOVA,
Fishers post hoc test ****p<0.0001; One way ANOVA, Tukey post hoc
***p<0.001.
51
T im e s p e n t in lig h t ( s e c )
A.
500
400
*
300
200
100
0
1
4
8
T im e (w e e k s )
B.
R e a r in g E v e n t s ( # )
25
SH AM
20
T IC
15
**
10
5
0
1
4
T im e (w e e k s )
8
Figure 2.2. Mice with TIC Had Anxiety- and Depressive-Like Behavior in the
Light-dark Box. Mice with TIC spent significantly less time in the light side of the
light-dark box at post injury week 8 compared with that of the sham animals (A).
Mice with TIC showed a decrease number of rearing events at post injury week 8
as compared to the sham mice (B). Naïve animals behavioral baselines are
indicated by the dotted line; n= 8-21, Two way ANOVA Fishers post hoc test
**p<0.01,*p<0.05.
52
D u r a t io n ( s e c )
A.
125
**
SH A M - STAR TLE
T IC - S T A R T L E
100
75
*5 0
25
0
1
4
8
T im e (w e e k s )
B.
T r a n s it io n ( # )
5
***
4
*
3
2
1
0
1
4
8
T im e (w e e k s )
C.
H e a d D ip s ( # )
15
10
5
0
1
4
8
T im e (w e e k s )
Figure 2.3. Acoustic Disturbance Affected Mice with TIC in the Elevated
Plus Maze. Mice with TIC spent significantly more time in the open arms post
injury week 1 compared with that of the sham animals (A). Mice with TIC showed
a significantly more number of transitions into the open arms at post injury week
53
1 and 4 as compared to the sham mice (B). Head dips into the open arms were
not significantly from mice with TIC compared to that of sham mice (C). Naïve
animals behavioral baselines are indicated by the dotted line; n= 8-11, Two way
ANOVA Fishers post hoc test ***p<.001, **p<0.01,*p<0.05.
54
B.
R e a r in g E v e n t s ( # )
500
400
*
300
350
R e a r in g T im e ( s e c )
A.
200
100
0
1
4
SH AM
300
T IC
250
*
200
150
100
50
0
8
1
T im e (w e e k s )
4
8
T im e (w e e k s )
C.
D.
500
**
2500
R e stin g T im e (s e c )
A c t iv e T im e ( s e c )
3000
2000
1500
1000
500
**
400
300
200
100
0
0
1
4
1
8
4
F.
2000
*
1500
6000
B r o k e n B ea m s (# )
D is t a n c e T r a v e le d ( c m )
E.
1000
500
0
1
4
8
T im e (w e e k s )
T im e (w e e k s )
5000
4000
3000
2000
1000
0
8
1
T im e (w e e k s )
4
8
T im e (w e e k s )
H.
1200
3000
1000
2500
T im e ( s e c )
T im e ( s e c )
G.
800
600
400
2000
1500
1000
500
200
0
0
1
1
4
8
4
8
T im e (w e e k s )
T im e (w e e k s )
Figure 2.4. Mice with TIC Injury Showed Decreased Exploratory Activity in
Open Field. Mice with TIC had a significantly fewer rearing events and rearing
compared to sham mice post injury week 8 (A&B). Mice with TIC had
significantly less active time and increased resting time post-op week 8
compared with that of sham mice (C&D). Mice with TIC traveled less overall
distance post-op week 8 compared with that of sham mice (E). No significant
55
difference was detected in beam breaks (F), central (G) or peripheral (H) time
duration. N=8-19, Two-way ANOVA, Fishers post hoc test, **p<0.01,*p<0.05.
56
CHAPTER THREE
RAPID EFFECTS OF PPAR-AGONISTS ON ATTENUATION OF
TRIGEMINAL PAIN
3.1. Introduction
Trigeminal neuropathic pain is an orofacial pain condition characterized by
continuous aching and burning sensation caused by trigeminal nerve
damage(Zakrzewska 2013). Dental procedures or trauma can cause trigeminal
peripheral nerve injury and inflammation, but in some cases, the cause is
unknown. Clinicians most often resolve treating this continuous trigeminal
neuropathic
pain
with
inadequate
anticonvulsants
and
antidepressant
((Asmundson & Katz 2009; De Leeuw 2008; Robinson et al 2009; Roditi et al
2009). Therefore, there is a great need for discovery of new drug targets.
Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor
with three isoforms: alpha, beta/delta and gamma. PPAR is widely expressed in
adipose, liver, cardiac, endometrial stromal cells, immune cells, neurons, and
glia of the peripheral and central nervous system (Cimini et al 2005a; Cimini et al
2005b; Cristiano et al 2005; Cullingford et al 2002; Gray et al 2012; Li et al 2010;
McKinnon et al 2012; Park et al 2007; Sarruf et al 2009). After ligand-activation,
the PPAR transcription factors of the nuclear hormone receptor superfamily plays
a major regulatory role in energy homeostasis and metabolic function (Michalik &
Wahli 2006). These receptors form a heterodimer with retinoid X receptor (RXR)
controlling gene expression of PPAR response elements (PPRE) on DNA
57
(Berger & Moller 2002; Moore et al 2001; Sanz et al 2012; Szanto et al 2004). In
particular, the activation of the peroxisome proliferator-activated receptor isoform
gamma (PPARγ) has shown to have multiple downstream effects.
PPARγ is activated by endogenous lipids or by thiazolidinediones, such as
rosiglitazone and pioglitazone (PIO), which is FDA approved for the treatment of
type 2 diabetes. These agonists have been shown to regulate fatty acid
metabolism (Nagashima et al 2005; Szanto et al 2004; Willson et al 2001).
However, more recent studies suggest PPARγ activation plays a role in another
major pathway that suppresses neuroinflammatory mediators, such as NF-κB
(nuclear factor kappa-light-chain-enhancer of activated B cells), thereby
decreasing microglial activation and certain cytokines such as TNF-α (tumor
necrosis factor alpha) and IL-6 (Interleukin 6) (Berger & Moller 2002; Combs et al
2000; Maeda & Kishioka 2009; Sadeghian et al 2012). As well as showing a
reduction in paw edema after capsaicin injection, PPARγ activation also reduces
mechanical allodynia and thermal hyperalgesia in the sciatic nerve injury animal
model (Fehrenbacher et al 2009; Ghosh et al 2007; Maeda & Kishioka 2009;
Morgenweck et al 2010; Morgenweck et al 2013; Park et al 2007). Furthermore,
PPARγ has been shown to be upregulated in Schwann cells after nerve injury
(Cao et al 2012; Zhang et al 2010). Other studies have found that the PPARγ
receptor is upregulated specifically within two weeks after an optic nerve injury in
a mouse and then decreases down to normal levels for later time points (Zhu et
al 2013).
58
Although PPARγ activation has been highly implicated in decreasing
specific types of neuropathic and inflammatory pain, the effects of PPARγ
activation on trigeminal pain has never been studied. Although, Moreno et al.
indicated PPAR to be in the trigeminal nucleus (Moreno et al 2004), but no one
has explored the function of this receptor in the Trigeminal Brainstem Sensory
Nuclear Complex (TBSNC) which is composed of four nuclei name rostral to
caudal: main sensory nucleus (principal nucleus 5), spinal trigeminal oralis,
spinal trigeminal interpolaris, and spinal trigeminal caudalis (De Leeuw 2008;
Sessle 2000). The TBSNC consists of second order neurons that relay tactile
and painful stimulation to the thalamus which then transmit the signals to the
sensory cortex (layer IV) corresponding to the relevant head/neck/facial region
making up the trigeminothalamic pathway (DaSilva & DosSantos 2012; De
Leeuw 2008; Sessle 2000). Therefore, the TBSNC is vital to trigeminal
nociception transmission.
The aim of this current study is to determine the role of PPARγ in trigeminal
neuropathic pain utilizing our novel Trigeminal Inflammatory Compression (TIC)
injury model in mice (Ma et al 2012a). Our hypotheses are that 1) PPARγ is more
immunoreactive in the mice with TIC injury and 2) PIO attenuates trigeminal TIC
injury pain dependent on PPARγ activation. We evaluated: 1) PPARγ
immonoreactivity
in
the
TBSNC
with/without
TIC
injury,
2)
systemic
administration of PIO, PPARγ agonist, in the mice with TIC injury to assess the
attenuation of trigeminal pain, and 3) systemic administration of PPARγ
59
antagonists to better determine whether PIO acts through PPARγ dependent
pathways in the attenuation of pain.
3.2.
Materials and Methods
3.2.1. Animals
See chapter 2; section 2.2.1.
3.2.2. Trigeminal Inflammatory Compression (TIC) Surgery
See chapter 2; section 2.2.2.
3.2.3. Assessment of Mechanical Allodynia on the Mouse Whisker Pad
See chapter 2; section 2.2.3.1.
3.2.4. Immunohistological Study
Mice with TIC, 3 weeks post injury and aged matched naïve mice were
anesthetized with isoflurane and decapitated. This early time point was based on
previous literature that indicated PPARγ changes occur at earlier time points
(Cao et al 2012; Zhu et al 2013). The brainstem was dissected and then placed
in 4% paraformaldehyde in 0.1 M phosphate buffer solution (PB, pH 7.4) for 42
hours. This was followed by a 24 hour soak in 30% sucrose in PB. The
brainstems were then embedded in OCT Compound (Tissue-Tek, Sakura,
Torrance, CA) and sectioned with a cryostat at the thickness of 40 microns and
60
sequentially placed in 24-well plates filled with Ethylene Glycol based anti-freeze
solution, stored at -20°C for immunohistological study. To insure the integrity of
the stain and control for variability, all tissues were simultaneously processed for
staining on the same day. On the day of immunostaining, the tissues were
washed with 0.1M PBS (pH 7.4) and pretreatment with 3% hydrogen peroxide in
0.1M PBS (pH 7.4) for 15 min to destroy endogenous peroxidase activity in
erythrocytes. Tissues were then blocked using 0.5% Triton X-100 in PBS (15min)
to permeabilize cell membranes and reduce cell surface tension to increase the
antibody penetration. The 5% normal goat serum in the PBS (40min) blocked
nonspecific antigen-antibody combinations. Sections were incubated overnight at
4°C with rabbit polyclonal anti-PPAR-gamma IgG (1st. antibody) (1:6000 dilutions;
H-100 Santa Cruz Biotechnology, Dallas, TX). The succeeding day, the sections
were incubated at room temperature with a secondary bioatinylated goat antirabbit IgG (1:200; company, place) for 40 minutes. The sections were then
incubated with avidin-biotin complex (ABC) reagent for 40 minutes. Finally the
antibody-antigen interaction was visualized via a peroxidase-catalyzed reaction.
After mounting the sections to gel-coated Super Plus glass slides, they were
allowed to air dry for at least 4 hours before they were dehydrated through
graded ethanol and xylene. Then the slides were cover-slipped using Permount
mounting medium (Fishers Scientific, Waltham, MA). The slides were imaged
using Nikon E1000 microscope (Nikon Instruments, Inc., Melville, NY) equipped
with a Nikon DXM1200F digital camera and the Act-1 Program.
61
3.2.5. Image Analysis
The immunostaining intensity of the trigeminal brainstem sensory nuclear
complex (main sensory, oralis, interpolaris, and caudalis) was analyzed (n=3; 912 tissues/animal) using ImageJ (1.46, NIH). Each subnuclei of the trigeminal
brainstem sensory nuclear complex was identified as a region of interest and
analyzed for mean fluorescent intensities. To identify PPARγ immunoreactivity
differences, each subnucluei of the complex was analyzed separately. The mean
fluorescent intensities of the trigeminal dorsal horn were measured in mice with
TIC injury and then compared to that in naïve mice
3.2.6. Drug Preparation and Administration
PPARγ agonist, pioglitazone, was dissolved in normal saline followed by
30 seconds of vortex. Then the solution was sonicated for 20 minutes before use.
Benzafibrate, PPARα agonist, was dissolved in 1% carboxymethylcellulose and
was then vortexed for 30 seconds. Fenofibrate, PPARα agonist; GW0742,
PPARβ/δ agonist and GW9662, PPARγ antagonist were dissolved in 10%
dimethyl sulfoxide (DMSO) and vortexed for 30 seconds before administration.
All drugs were fresh prepared on the day just before administration.
Drugs were administered after mechanical allodynia was confirmed at
least 8 weeks post operation to observe the efficiency of each drug in a chronic
neuropathic pain condition. All doses were chosen based on drug safety, and
drug efficacy in previous studies (Fehrenbacher et al 2009; Feinstein et al 2005;
62
Gross et al 1999; Hamano et al 2011; Maeda & Kishioka 2009; Morgenweck et al
2010; Morgenweck et al 2013; Oliveira et al 2007; Paterniti et al 2012)
Given systemically with the following doses: pioglitazone at doses 100
mg/kg and 300 mg/kg (<10ml/kg volume) was injected intraperitoneal (<10 ml/kg
volume) and 600 mg/kg was given oral gavage (at the volume of <10 ml/kg).
Lower doses of pioglitazone (≤100 mg/kg) have been reported in previous
studies, but when no effect was observed at 100 mg/kg, doses were increased
accordingly. For a mouse, the reported LD50 of PIO given systemically ranges
from 181 mg/kg-1200 mg/kg, therefore the 600 mg/kg dose was administered
orally (United States Pharmacopeial Convention, 2013)].
PPARα agonists, benzofibrate (100 mg/kg p.o.; LD50 500 mg/kg) and
fenofibrate (200 mg/kg i.p.; LD50 1200 mg/kg), and PPARβ/δ agonist, GW0742
(1 mg/kg and/or 6 mg/kg i.p.) were given systemically to serve as PPAR
activation controls. The LD50 of GW0742 has not been reported.
In a separate cohort of mice, the PPARγ antagonist, GW9662 (30 mg/kg
i.p), was injected 30 minutes before the PPARγ agonist, pioglitazone (300 mg/kg
i.p.) was given. This dose of PIO was chosen because it had the maximum effect
on the elevation of mechanical threshold in TIC injury animals. GW9662 was
employed to block PIO from binding to PPARγ to see if the effect of PIO is
specific via activation of PPARγ (Feinstein et al 2005; Lea et al 2004; Maeda &
Kishioka 2009).
During each drug testing, 50% mechanical threshold was measured at
time points of 0, 0.5, 1, 2, 3, 4 hours post inject on the ipsilateral whisker pad
63
only. The 600 mg/kg oral dosing of PIO was measured out until 6 hours because
an attenuated effect was observed starting at the 4th hour post injection time
point. To increase animal “n” number in the treatment groups for these
experiments, mice were tested with these drugs using the Latin square type
crossover method with at least 1 week interval between each drug testing and
allow sufficient time for the effect of a previous treatment to wear off. Vehicle for
the PIO study was normal saline. For all other drugs, 10% DMSO was
administered to the mice with TIC injury to serve as a vehicle. One experimenter
was blinded to the drugs given to the animals for each experiment.
3.2.7. Statistical Analysis
The data were expressed as means  S.E.M. The GraphPad Prism 6
statistical program was used for data analysis (Graph Pad Software, Inc., La
Jolla, CA). All behavioral data including drug studies were analyzed using a TwoWay ANOVA with a Fishers post hoc test; Histological studies were analyzed by
a Two-Way ANOVA with a Tukey post hoc test; (where is appropriate) p≤0.05 is
considered significant.
64
3.3.
Results
3.3.1 Mice with TIC Injury Demonstrated Unilateral Whisker Pad Mechanical
Allodynia
The 50% baseline mechanical threshold was initially the same on both
side of the whisker pad of mice with/without TIC (3.51 ± 0.18 g for the left; 3.74 ±
0.45g for the right). The mice with TIC injury experienced unilateral mechanical
allodynia on the ipsilateral whisker pad within one week post operation lasting
until the euthanasia day (week 14 post-injury). The mean 50% mechanical
threshold of the ipsilateral whisker pad for the mice with TIC injury was 0.24 ±
0.92 g making it statistically significant compared to that on the contralateral
whisker pad (3.51 ± 0.18 g; n=8; p<0.0001, two-way ANOVA, Fishers post hoc
test, Figure 3.1). In contrast, the sham operation control mice did not show any
changes in the mechanical threshold after the surgery. Compared to sham
operation control mice, the mean mechanical thresholds of the ipsilateral whisker
pad of the mice with TIC injury was statistically significant (0.24 ± 0.92g vs. 3.51
±0.36g; n=8; p<0.0001, two-way ANOVA, Fishers post hoc test).
65
3.3.2. The Effect of PPAR Agonists on Mechanical Allodynia of Mice with TIC
Injury
3.3.2.1.
PPARγ
Agonist,
Pioglitazone,
Attenuated
Mechanical
Allodynia in Mice with TIC Injury
At 8 weeks post operation, pioglitazone (300 mg/kg i.p.) effectively
attenuated mechanical allodynia of the ipsilateral whisker pad, the 50%
mechanical threshold from 0.24 ± 092 g before drug treatment increased to 1.61
± 0.54 g at hour 1 peaking at hour 2 (2.72 ± 0.74 g) lasting for 3 hours (2.33 ±
0.98 g; Figure 3.2A). This was statistically significant from those of the mice with
TIC injury treated with saline injection (2.72 ± 0.74 g vs 0.10 ± 0.04 g; two-way
ANOVA, Fishers post hoc test, ****p<0.0001; n=3-7). The p.o. administration of
pioglitazone (600 mg/kg p.o.) also attenuated mechanical allodynia in the mice
with TIC injury (5hr: 0.87 ± 0.32 g; 6hr: 0.92 ± 0.45 g; two-way ANOVA, Fishers
post hoc test, *p<0.05; n=3-7) compared that to the saline treated mice with TIC
injury (0.03 ± 0.00 g; two-way ANOVA, Fishers post hoc test, *p<0.05; n=3-7)
which is significantly different at 5 and 6 hours post injection. However, this dose
was not as effective as the 300 mg/kg i.p. of pioglitazone. The 100 mg/kg i.p.
dose of pioglitazone did not have any effect on the mice with TIC injury
compared to that of the saline treated mice (0.56 ± 1.40 g vs 0.10 ± 0.04 g). The
300 mg/kg dose elicited hypothermic side effects that were most likely an
indication that the dose was too high. However, the 100 mg/kg and 600 mg/kg
doses did not elicit any observable overt side effects. These results
66
demonstrated that PPARγ receptor is a key player in alleviating whisker pad
mechanical allodynia in the mice with TIC injury.
3.3.2.2.
PPARβ/δ
Agonist
Moderately
Attenuated
Mechanical
Allodynia in the Mice with TIC Injury
Allodynia in the Mice with TIC Injury The administration of GW0742,
PPARβ/δ agonist (6 mg/kg i.p.), partially attenuated mechanical allodynia in mice
with TIC injury compared to that of the mice with TIC injury treated with vehicle.
The effect reached peak at hour 2 post injection (1.59 ± 0.55 g vs. 0.06 ± 0.02 g;
two-way ANOVA, Fishers post hoc test; ****p<0.0001, n= 4-6; Figure 3.2B). The
GW0742 administration of (1 mg/kg i.p.) provided no attenuation of mechanical
allodynia (0.51 ± 1.31 g). These results showed that the activation of PPARβ/δ
isoform play some role in the control of mechanical allodynia induced by the TIC
injury.
3.3.2.3.
PPARα Agonist Had No Effect on the Mice with TIC Injury
Two PPARα agonists were used in this experiment. The first one is
benzafibrate which is a pan-PPAR agonist with the highest affinity for the alpha
subunit. Benzafibrate at 100 mg/kg i.p. injection, had no effect on mechanical
allodynia in mice with TIC injury (treated: 0.55 ± 1.34 g vs. vehicle: 0.71 ± 1.52 g;
two-way ANOVA, Fishers post hoc test; p>0.05, n= 3-6; Figure 3.2C). The
second and more specific PPARα agonist, fenofibrate, was administered at 200
mg/kg i.p., no attenuation effect on mechanical allodynia effect (0.56 ± 1.28 g)
was observed with this drug at this particular dose. These results demonstrated
67
that activation of PPARα at these doses do not contribute to the attenuation of
the mechanical allodynia in the mice with TIC injury.
3.3.2.4.
PPARγ Antagonist, GW9662, Blocked Analgesic Effects of
Pioglitazone
GW9662, a potent antagonist of PPARγ, at the dose of 30 mg/kg (i.p.)
successfully blocked the actions of pioglitazone (300 mg/kg i.p.) in alleviating
mechanical allodynia in the mice with TIC (GW9662+PIO: 0.53 ± 1.29 g vs. PIO
only: 1.55 ± 1.41 g; two-way ANOVA, Fishers post hoc test, N=4-7; ****p<0.0001,
Figure 3.2D). These results provide evidence that PIO is acting through a
PPARγ dependent mechanism to attenuate mechanical allodynia. Due to the fact
that mechanical allodynia in the mouse whisker pad was attenuated post 3 hours
after PIO injection, support is given to the theory that PPARγ is working through
nongenomic mechanisms to attenuate trigeminal pain.
3.3.3. PPARγ Was More Immunoreactive in the TBSNC of Mice with TIC injury
The entire trigeminal brainstem sensory nuclear complex (TBSNC)
(composed of the main sensory trigeminal nucleus, and spinal trigeminal oralis,
spinal trigeminal interpolaris, and spinal trigeminal caudalis was sectioned and
stained for PPARγ immunoreactivity. The spinal trigeminal dorsal horn (sp5)
sections were imaged and analyzed for PPARγ immunoreactivity (mean
intensities) in mice three weeks after TIC injury and along with naïve controls.
Although the PPARγ positive neurons were expressed in the TBSNC of both
68
mice with TIC and naïve mice, the PPARγ-like immunoreactivity greatly
increased in the injured animals. Figure 3.3 depicts the mean intensities of the
immunoreactivity of PPARγ throughout the trigeminal brainstem complex in the
mice with TIC injury compared to that of the naïve mice. There was a significantly
higher optical intensity for PPARγ immunoreactivity in the TBSNC of the mice
with TIC injury. The main sensory nuclei (85.65 ± 19.37), spinal trigeminal oralis
(65.43 ± 12.68), and spinal trigeminal caudalis (93.59 ± 27.62) on the ipsilateral
side of TIC injury was compared to that of the naïve mice (main sensory nucleus:
67.21 ± 14.01; sp5 oralis: 46.42 ± 18.46; sp5 caudalis: 71.76 ± 17.31; p<0.05;
two-way ANOVA, Fishers post hoc test, n=3 with 9-12 sections/animal). The
immunostaining intensity in the spinal trigeminal interpolaris of the mice with TIC
injury (75.61 ± 17.48) did not show significant difference from those of the naïve
mice (65.64 ± 15.78). However, PPARγ immunoreactivity in the spinal trigeminal
caudalis of the mice with TIC injury was greater than that of the rest subnuclei of
the trigeminal brainstem complex and significantly different from the spinal
trigeminal oralis (p<0.01) and the spinal trigeminal interpolaris (p<0.05; two-way
ANOVA, Fishers post hoc, n=3 with 9-12 sections/animal). Furthermore, there
was a bilateral increase in PPARγ immunoreactivity only in the spinal oralis (TIC
contralateral: 67.22 ± 10.89).
Figure 3.4 shows tissue slices of the entire TBSNC in a naïve mouse
compared to that of a mouse with TIC injury. Sample tissues representing each
level of the TBSNC from a mouse with TIC injury are depicted in figure panels BD (x20).
69
Figure 3.5 depicts the ipsilateral sp5 interpolaris in the mice with TIC
injury compared to that in naïve mice. The white arrows indicate the projection of
whisker barrels of sp5 that have been shown to correlate to the receptive fields
on whisker pads of the mice (Zembrzycki et al 2013). As indicated at 20x image,
this is primarily where positive PPARγ neurons were located indicated by the
black arrows. There also appears to be glial nuclei stained for PPARγ consistent
with previous findings (Cao et al 2012; Maeda & Kishioka 2009; Sadeghian et al
2012). Although PPARγ immunoreactivity also appears in the TBSNC of naïve
mice, the mean staining intensity was much less compared to that of the mice
with TIC injury.
3.4.
Discussion
This study determined that the nuclear receptor, peroxisome proliferator
receptor-gamma isoform (PPARγ), plays a significant role in trigeminal pain
transmission. Histology revealed that 3 weeks after the Trigeminal Inflammatory
Compression (TIC) injury, compared to the other subnuclei of the trigeminal
brainstem complex, a most intense PPARγ immunoreactivity appeared in the
spinal trigeminal caudalis where the primary pain fibers actually synapse onto.
Systemic administration of a PPARγ agonist, pioglitazone (PIO), attenuated the
mechanical allodynia in the mice with TIC injury at doses of 300 mg/kg i.p. and
600 mg/kg p.o. However, 100 mg/kg of PIO (i.p.) had no effect on the mice with
TIC injury. Furthermore, these studies revealed that administering a PPARγ
antagonist, GW9662 (30 mg/kg i.p.) prior to the optimal dose of PIO (300 mg/kg
70
i.p.) blocked the analgesic effect of PIO indicating that PIO is acting through a
PPARγ mechanism. Additionally, the PPARα agonists, benzafibrate and
fenofibrate, had no effect on the allodynic mice. However, the PPARβ/δ agonist,
GW0742 had a minimal attenuation of allodynia effect to the mice with TIC injury.
Taken together, these results confirm PPAR’s role in trigeminal pain
transmission.
Based on these findings, PPARγ and PPARβ/δ function to inhibit pain
transmission once activated. However, PPARα does not appear to be a key
player in trigeminal neuropathic pain. Some controversial studies show that
PPARα activation have an inhibitory effect on nociception after nerve injury or
inflammation (Benani et al 2004; LoVerme et al 2006; Maeda & Kishioka 2009;
Oliveira et al 2007). No study has ever reported PPARα activation eliciting an
analgesic effect in trigeminal neuropathic pain. However, in this study, the doses
of the two PPARα agonists could have been increased to possibly provide an
analgesic effect. Future studies should be conducted to observe if higher doses
comparable to pioglitazone will attenuate the mechanical allodynia observed in
the mice with TIC injury. On the other hand, PPARβ/δ agonists have been
reported to attenuate inflammatory pain (Gill et al 2013; Hall et al 2008), but little
about this receptor isoform has been reported. The biological role of PPARβ/δ
has remained elusive due, in part, to its broad tissue expression and the lack of
good chemical tools with which to study its physiological function. Future studies
could be conducted to observe PPARβ/δ immunoreactivity in along the trigeminal
dorsal horn.
71
Unlike PPARβ/δ, PPARγ has been identified more thoroughly. Upon
PPARγ activation, it will inhibit not only inflammation, but also neuropathic pain
after an injury (Fehrenbacher et al 2009; Ghosh et al 2007; Maeda & Kishioka
2009; Morgenweck et al 2010; Morgenweck et al 2013; Park et al 2007).
Furthermore, little is known about PPARγ in the trigeminothalamic pathway.
Moreno and colleagues (Moreno et al 2004) confirmed PPARγ was present in the
dorsal horn of the brainstem and spinal cord, and Maedo and colleagues (2005)
(Maeda et al 2005) confirmed Moreno’s findings by using immunohistochemistry
to identify PPARγ in the sciatic nerve, dorsal root ganglia, and dorsal horn
supporting PPAR’s representation along the nociceptive pathway.
This study was not only the first to identify PPARγ immunoreactivity
throughout the trigeminal brainstem sensory nuclear complex with/without
trigeminal nerve injury, but this study also was the first study to demonstrate that
PPARγ activation attenuates trigeminal hypersensitivity in the TIC injury model.
This study revealed that PPARγ immunoreactivity increases three weeks after
the Trigeminal Inflammatory Compression (TIC) injury consistent with other
findings in which PPARγ becomes upregulated within weeks after a nerve injury
(Cao et al 2012; Zhu et al 2013). Additionally, the results of this study support
previous studies demonstrating PPARγ’s presence in neuronal and glial cells
(Maeda & Kishioka 2009). Although this study identified PPARγ immunoreactivity
in putative neurons, future double-labeling experiments in the TBSNC with
PPARγ, neuronal, and glial markers (neuN, IB-4, OX-42, etc.) would serve as
72
more conclusive evidence of PPARγ’s presence in neurons and/or glial cells,
supporting its role in neurogenic inflammation.
Furthermore, PPARγ activation has been shown to transrepress NF-κB
thereby downregulating pro-inflammatory cytokines
such as IL-6 and TNF-α
(Berger & Moller 2002; Combs et al 2000; Maeda & Kishioka 2009; Sadeghian et
al 2012; Scholz & Woolf 2007). Previous studies have even reported that PPARγ
deficient mice are more vulnerable to inflammatory diseases (Adachi et al 2006;
Cuzzocrea et al 2004; Straus & Glass 2007). In our previous study (Ma et al
2012a), we demonstrated microglial activation at the trigeminal dorsal horn of
mice with TIC injury. Therefore, one possible reason for the increase in PPARγ
immunoreactivity at the TBSNC observed in the mice with TIC injury could be the
inflammatory response occurring in the trigeminal dorsal horn. The upregulation
of PPARγ at the trigeminal nucleus would be a way to combat the neural immune
system and downregulate the pro-inflammatory cytokines once PPARγ is
activated.
Interestingly, PPARγ immunoreactivity was most intense in the spinal
trigeminal caudalis of the mice with TIC injury while barely detectable in naïve
mice. Since the spinal trigeminal caudalis has been identified as the primary
nucleus for nociceptive signaling (De Leeuw 2008; Sessle 2000), the
upregulation might partially explain why the PPARγ receptor agonist, PIO, has
analgesic effects on mechanical allodynia in mice with TIC injury. With the
upregulation of PPARγ, a stronger anti-inflammatory cell signaling cascade could
be initiated once PIO binds to its receptor.
73
However, there is much debate about the actions of PIO. Some scholars
believe that PIO activates PPARγ to induce transcription while others believe
there are PPARγ transcription independent mechanisms that occur to decrease
allodynia (Fehrenbacher et al 2009; Feinstein et al 2005; Lea et al 2004; Maeda
& Kishioka 2009; Thal et al 2011). Still, others suspect and have possibly
demonstrated that PIO can act on other intracellular receptors such as acting on
a mitochondrial membrane protein known as mitoNEET (Yonutas & Sullivan
2013). However, in present study, when PPARγ antagonist, GW9662, blocked
PIO’s analgesic actions, it was then concluded that PIO attenuates trigeminal
nociception by acting through PPARγ. More studies need to be conducted to
elucidate the action of PIO non PPARγ dependent pathways.
3.5.
Conclusion
Overall this novel study determined that PPARγ activation by PIO plays the
most potent role in attenuating mechanical allodynia in the mice with TIC injury.
This experiment was the first to map PPARγ in the trigeminal brainstem sensory
nuclear complex as well as show an increase in PPARγ immunoreactivity after
three weeks post injury. Taken together, these studies provide a new target,
PPARγ, for attenuation of trigeminal pain which raises the possibility for
repurposing the FDA approved diabetic therapeutic drug, PIO, for the treatment
of patients suffering from orofacial pain.
74
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
M e c h a n ic a l A llo d y n ia
7
S h a m Ip s ila te ra l
6
S h a m C o n tra la te ra l
5
T IC Ip s ila te ra l
T IC C o n tra la te ra l
4
3
2
1
****
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
T im e ( w e e k s )
Figure 3.1. Mice with TIC Injury Developed Unilateral Mechanical Allodynia
on the Ipsilateral Whisker Pad. The 50% mechanical threshold on whisker
pads of the mice with TIC injury and the sham mice were measured bilaterally for
detecting mechanical allodynia. The 50% mechanical threshold on the ipsilateral
whisker pad of mice with TIC injury was dramatically decreased within one week
of injury lasting until euthanasia, week 14. The mechanical threshold on
contralateral whisker pad of the mice with TIC injury was unaffected by the
surgery. The mechanical threshold on the whisker pads of the sham operation
mice did not change (n=9/group; ****p<0.0001, two-way ANOVA, Fishers post
hoc test).
75
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
A.
4 .5
V e h ic le (s a lin e )
4 .0
****
3 .5
2 .5
2 .0
1 .5
T IC
*
*
5
6
1 .0
0 .5
0 .0
L
In
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
P io g litz a o n e (6 0 0 m g /k g p .o .)
****
B
ti
c
je
o
n
0
.5
1
2
3
4
T im e ( h )
5
V e h ic le ( 1 0 % D M S O )
4
G W 0 7 4 2 (1 m g /k g )
G W 0 7 4 2 (6 m g /k g i.p .)
3
****
2
T IC
1
0
B
L
In
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
P io g lita z o n e ( 3 0 0 m g /k g i.p .)
3 .0
B.
je
ti
c
o
.5
n
5
1
2
3
4
T im e ( h )
V e h ic le ( 3 % D M S O )
B e z a f ib r a te ( 1 0 0 m g /k g p .o .)
4
F e n o fib r a te (2 0 0 m g /k g i.p .)
3
2
T IC
1
4
3
2
1
0
n
In
je
c
ti
o
B
D.
.5
0
L
V e h ic le (s a lin e )
4 .5
P IO (3 0 0 m g /k g )
4 .0
G W 9 6 6 2 (3 0 m g /k g ) + P IO (3 0 0 m g /k g )
**** ****
3 .5
3 .0
2 .5
****
2 .0
1 .5
T IC
1 .0
0 .5
T im e ( h )
76
4
3
2
1
.5
je
c
ti
o
B
n
L
0 .0
In
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
T im e ( h )
0
C.
P io g lita z o n e ( 1 0 0 m g /k g i.p .)
****
Figure 3.2. Pioglitazone (PIO) Attenuate Mechanical Allodynia in the Mice
with TIC Injury. Hypersensitivity was blocked by specific PPARγ antagonism
with (A) PIO rapidly elevating the 50% mechanical threshold in the mice with TIC
at higher doses (300 mg/kg and 600 mg/kg), but was ineffective at 100 mg/kg
(n=3-7). (B) GW0742, PPARβ agonist, attenuated mechanical allodynia in the
mice with TIC at a dose of 6 mg/kg, but it still not as effective as PIO (n=4-6). (C)
PPARα agonists, benzafibrate and fenofibrate, were not effective in alleviating
mechanical allodynia in the mice with TIC (n=3-6). (D) PPARγ antagonist,
GW9662, blocked the effects of PIO at a dose of 30 mg/kg (n=4-7). *p<0.05,
****p<0.0001; two-way ANOVA, Fishers post hoc test.
77
#
##
125
N a iv e Ip s ila t e r a l
*
M e a n In t e n s ity
N a iv e C o n t r a la t e r a l
*
100
T IC Ip s ila t e r a l
* *
75
T IC C o n t r a la t e r a l
50
25
0
M
a
in
S
e
n
s
o
ry
O
ra
li
s
In
te
rp
o
la
ri
s
C
a
u
d
a
li
s
S p 5 N u c le i (R o s tr a l t o C a u d a l)
Figure 3.3. PPARγ Immunoreactivity Increased in TBSNC of Mice with TIC
Injury. The Trigeminal Brainstem Sensory Nuclear Complex (TBSNC) was
stained for anti-PPARγ antibody in the mice with TIC injury and naïve mice. The
images show the individual subnuclei of the TBSNC (main sensory/principle five,
spinal oralis, spinal interpolaris, spinal caudalis) and PPARγ immunoreactivity in
each
nucleus.
The
mice
with
TIC
injury
had
an
increased
PPARγ
immunoreactivity in the main sensory, spinal oralis and spinal caudalis on the
ipsilateral side compared to those in the ipsilateral side of the naïve mice (n=3
with 9-12 sections/animal ;*P<0.05, two-way ANOVA, Fishers post hoc). The
spinal trigeminal nucleus oralis expressed bilateral immunoreactivity that was
significant greater than tissue from naïve mice (*P<0.05, two-way ANOVA,
78
Fishers post hoc). PPARγ immunoreactivity on the ipsilateral side of the mice
with TIC injury also was significantly higher in the spinal caudalis compared to
that of spinal trigeminal oralis and interpolaris (#p<.05, ##p<0.01, two-way
ANOVA, Fishers post hoc).
79
A
Naive
B
TIC
Rostral
Pr5
C
Or5
Inter5
D
Caud5
Caudal
E
80
Figure 3.4. PPARγ Immunoreactivity Localized Throughout the Trigeminal
Brainstem Sensory Nuclear Complex in Mice with TIC Injury. (A) The image
depicts the TBSNC rostral to caudal in naïve mice compared to mice with TIC.
The subnuclei are indicated as follows (B) principal 5 (Pr5), (C) spinal
subnucleus oralis (Or5), (D) spinal subnucleus interpolaris (Inter5), and (E) spinal
subnucleus caudalis (Caud5).
81
A
NAIVE
B
100μm
50μm
C
TIC
D
100μm
50μm
Figure 3.5. PPARγ Immunoreactivity at the Spinal Trigeminal Nucleus
Interpolaris in Sham Mice vs. Mice with TIC Injury. The upper panel depicts
the ipsilateral side of the naive mice at 10x (A) and 20x (B). The lower panel
depicts the ipsilateral side of the mice with TIC at 10x (C) and 20x (D). The black
arrows indicate the PPARγ positive cells and the white arrows indicate the
projection of whisker barrels that correspond to the receptive fields on the mouse
whisker pad where most PPARγ positive cells were found. As shown by the
pictures, the staining intensity of PPARγ immunoreactivity was more intense in
the tissue from the TIC injured mice than that of the tissue from the naïve mice.
82
CHAPTER FOUR
LOW-DOSE COMBINATION OF PIOGLITAZONE AND D-CYCLOSERINE
ATTENUATED OROFACIAL PAIN BY IMPROVING MITOCHONDRIAL
DYSFUNCTION
4.1.
Introduction
Chronic trigeminal neuropathic pain is an orofacial pain condition
characterized by chronic aching and burning sensation sometimes overlaid with
sharp, electric-like shooting pain caused by trigeminal nerve damage. This injury
could be due to the compression of the trigeminal nerve by an arteriole pulsation
or could also be caused by a peripheral nerve injury initiated by dental trauma or
unknown cause (Burchiel 1993; Cruccu et al 1990; De Leeuw 2008; Devor et al
2002a; Devor et al 2002b; Jannetta 1967a; b; Love & Coakham 2001). This type
of injury and pain is very difficult to treat so new therapeutics are needed that
target key players in the ensuing cell stress that follows.
The chemiosmotic theory states that during the oxidation of mitochondrial
complexes, “free energy” in the form of protons (H+ ions) are released into the
intermembrane space creating the proton motive force (Sheinin et al 2001). In
healthy mitochondria, the protons will flow back through complex V (ATP
synthase) inducing ATP synthesis. However, a disruption to any specific complex
along the mitochondrial electron transport chain (mETC) will lead to one of the
following energy-dissipating pathways (EDP): 1) decreased electron transport
along the mETC, 2) increased proton concentration in the intermembrane space,
83
3) lowered ATP production, or 4) increased reactive oxygen species (Brand &
Nicholls 2011; Starkov 2008; Starkov et al 2004; Wallace & Starkov 2000). Cell
stress can often be triggered by mitochondrial dysfunction, referring to
any
energy dissipating pathway (EDP) decreasing mitochondrial bioenergetics
(Brand & Nicholls 2011; Starkov 2008).
Studies have indicated mitochondrial dysfunction is a major player not
only in cell stress, but also in the etiology of inflammatory and chronic
neuropathic pain that occurs after peripheral nerve injuries (Bouillot et al 2002;
Ferrari & Levine 2010; Joseph & Levine 2006; Kim et al 2004; Shin et al 2003;
Sui et al 2013). Joseph and Levine (2006) showed that after administering
inhibitors to the specific complexes of the mitochondrial electron transport chain
(mETC), mechanical allodynia was attenuated in animals with sciatic nerve injury
(Joseph & Levine 2006).
Other studies have shown that after spinal cord or brain injury,
mitochondrial dysfunction and oxidative stress also occur within 24 hours of the
injury (Sullivan et al 2007; Sullivan et al 2004a; Sullivan et al 2004b). However,
administration of a mild mitochondrial uncoupler, such as 2, 4-Dinitrophenol (2,4DNP), has been shown to protect the cortex from mitochondrial dysfunction and
reactive oxygen species (ROS) production in mice after spinal cord injury (Brand
& Esteves 2005; Mahmud et al 1996; Pandya et al 2007; Patel et al 2009). Mild
uncouplers, such as 2,4-DNP, decrease the proton concentration gradient in the
intermembrane space by creating protonophores in the mitochondrial membrane
to allow H+ ions to freely flow across the mitochondrial membrane into the matrix
84
without crossing through the ATP synthase. In naïve animals, this lowers ATP
production and alters the mitochondrial oxygen consumption. However, during
cellular stress, mild mitochondrial uncouplers have been shown to decrease the
production of mitochondrial ROS and increase bioenergetics (Brand & Esteves
2005; Jin et al 2004; Rolo & Palmeira 2006; Sullivan et al 2004b).
Although studies have shown that ROS are produced in the trigeminal
nucleus after peripheral nerve injury and that ROS play a major role in pain
transmission (Alp et al 2010; Viggiano et al 2004; Viggiano et al 2005; Viggiano
et al 2010), whether peripheral trigeminal nerve injury can induce mitochondrial
dysfunction have never been studied to date.
Furthermore, FDA approved drug, (R)-(+)-4-Amino-3-isoxazolidinone (Dcycloserine) (DCS), known under the same Seromycin ® (DCS capsules, USP,
250 mg) is a broad spectrum antibiotic used alternatively for tuberculosis. It is a
derivative of the naturally occurring amino acid serine and acts as a partial
agonist at the strychnine insensitive glycine recognition site of the NMDA
receptor complex (Furukawa & Gouaux 2003; Hood et al 1989; Monahan et al
1989; Sheinin et al 2001). Binding of DCS to the NMDA complex enhances
glutamate activation and increases calcium influx, thus enhancing excitatory
neurotransmission (Heresco-Levy & Javitt 1998; Tomek et al 2013). However,
DCS when administered in higher doses has been shown to act as an NMDA
antagonist to reduce hypersensitivity in sciatic nerve injury in rats (Millecamps et
al 2007). Clinical trials showed that DCS is effective in the extinction of acquired
fear when used as an adjuvant to exposure therapy for anxiety disorders (e.g.
85
post-traumatic stress disorder, phobias, obsessive-compulsive disorder) (Davis
et al 2006; Heaton et al 2010; Norberg et al 2008). Only one clinical case study
to date has ever observed the anti-allodynic effects of DCS for alleviation of
chronic facial pain (Antal & Paulus 2011). Although DCS seems to be a
prospective new therapy for the treatment of orofacial pain, it has never before
been thoroughly tested in animals or a mechanism defined.
Pioglitazone (PIO), FDA approved, is a prescription drug (Actos) of the
class thiazolidinedione (TZD) with hypoglycemic (anti-hyperglycemic, antidiabetic) action to treat type 2 diabetes. Pioglitazone is a selective agonist of the
nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ).
However, studies have shown the PIO reduces hypersensitivity in the sciatic
nerve injury animal model (Fehrenbacher et al 2009; Ghosh et al 2007; Maeda &
Kishioka 2009; Morgenweck et al 2010; Morgenweck et al 2013; Park et al 2007).
Many have theorized that PIO is acting through PPARγ to decrease microglial
activation and oxidative stress (Collino et al 2006; Combs et al 2000; Sadeghian
et al 2012; Thal et al 2011). However, more recent data has shown that PIO is
also acting through a PPARγ independent mechanism on the mitochondria by
directly activating mitoNEET to decrease mitochondrial oxidative stress
(Geldenhuys et al 2014; Wiley et al 2007a; Wiley et al 2007b; Yonutas & Sullivan
2013). Recent studies in our laboratory have shown that PPARγ agonist,
pioglitazone (PIO), attenuates mechanical allodynia in the whisker pads of mice
after the Trigeminal Inflammatory Compression (TIC) injury primarily by a PPARγ
dependent pathway (see chapter 3). However, our data also suggest that since
86
the analgesic effect occurs within 2 hours, a PPARγ nongenomic mechanism is a
possibility. However, PIO’s actions in isolated mitochondria after a peripheral
trigeminal nerve injury have never been studied.
In this study, a trigeminal inflammatory compression (TIC) mouse model
was employed to investigate the effect of DCS or PIO, and DCS/PIO combination
on the relief of neuropathic nociception and anxiety associate with trigeminal
neuropathic pain. The effect of DCS/PIO combination on isolated brain
mitochondria after a peripheral trigeminal nerve injury was also explored.
87
4.2.
Materials and Methods
4.2.1. Animals
See Chapter 2; section 2.2.1.
4.2.2. Trigeminal Inflammatory Compression (TIC) Injury
See Chapter 2; section 2.2.2.
4.2.3. Behavioral Assays
4.2.3.1.
Detecting Mechanical Allodynia with von Frey Fiber Test
See chapter 2; section 2.2.3.1.
The assessment of mechanical allodynia after the drug administration was
only conducted on the ipsilateral whisker pad 8 weeks after injury. This time point
after injury was chosen since anxiety-and depression-like behavior develops 6-8
weeks after injury, as discussed in chapter 2 (Yalcin et al 2011). For one-time
injections, mechanical threshold was measured at 0, 0.5, 1, 2, 3, and 4 hours
drug injection, with hours 5, 6, 7, and 8 post injection evaluated if drug effect
persisted. For daily injections, the mechanical thresholds of the mice whisker
pads were measured once daily at the same testing time each day. This time
point was determined by the peak effect time point of the one-time injection, and
usually was between 2.5 - 3 hours after injection. One experimenter was blinded
to the drugs given at all times.
88
4.2.3.2.
Light-Dark Box Preference Testing
This test is used to observe anxiety-like behavior in the mice with TIC injury
(saline vs. drug treated). We have already determined that mice with TIC injury
developed an anxiety-and depressive-like behavior at the 8th week post injury
indicated by a prolonged duration of the occupancy in the light side of the box
(see chapter 2, section 2.2.3.4).
4.2.4. Drug Preparation and Administration
4.2.4.1. Drug Preparation
D-cycloserine (DCS), NMDA agonist/antagonist, was easily soluble in
normal saline (0.9% NaCl, 10 mg/ml). Pioglitazone (PIO), PPARγ agonist, was
dissolved in normal saline and vortexed for 30 sec before 20 minutes in the
sonicator. 2, 4-Dinitrophenol (2,4-DNP) was dissolved in 10% DMSO solution
followed by a 30 second vortex.
4.2.4.2. Drug Administration
For subcutaneous (s.c.) injections drug was administered at a volume of ≤
5 ml/kg/site using a sterile syringe with a 25G needle. For intraperitoneal (i.p.)
injection with 25G needle syringe, at a volume of ≤ 10 ml/kg (Turner et al 2011).
One experimenter was blinded to the drug treatments given for each experiment
described.
89
4.2.4.2.1. Experiment 1: Treatment of Mechanical Allodynia with
NMDA Receptor Agonist/Antagonist, DCS
The mice with TIC were given subcutaneous (s.c.) injections of the
following DCS doses in mg/kg (40, 60, 80, 100, 160, and 320 in 150 µl/mouse).
These doses were chosen based on previous published studies (Davis et al
2006; Kushner et al 2007; Lanthorn 1994; Millecamps et al 2007). All drugs were
administered at least 8 weeks after TIC injury since anxiety-like behaviors were
expected to develop 6- 8 weeks post the TIC injury (see chapter 2). The goal was
to observe the anti-anxiety effects of the drugs. Mechanical allodynia was
assessed every hour starting at 0 hour time point, for 4 hours. The vehicle control
mice with TIC injury received a subcutaneous injection of normal saline
(150µl/mouse)
At least 8 weeks after the TIC injury, mice started receiving daily
subcutaneous injections of a low dose of DCS (40 mg/kg, 60 mg/kg, and 80
mg/kg). In parallel to the mice with TIC injury, sham mice were also given the
same injections to observe the DCS effects as control. Mechanical threshold was
measured daily 2.5-3 hours after injection. On the 7th day, the mice receiving the
80 mg/kg dose went through the light/dark box preference testing. The control
mice with TIC injury and sham mice received a daily subcutaneous injection of
normal saline (150µl).
90
4.2.4.2.2. Experiment 2: One-time Injection of Combination of
DCS/PIO
In a separate cohort of animals, at least 8 weeks after TIC injury, mice
received one of the three injections 1) PIO, 100 mg/kg i.p., 2) DCS, 80 mg/kg
s.c., or 3) Combination injection: PIO, 100 mg/kg i.p. injection quickly followed by
DCS, 80 mg/kg s.c. injection. As mentioned in chapter 3, the reported LD50 of
PIO given systemically in a mouse ranges from 181 mg/kg-1200 mg/kg (United
States Pharmacopeial Convention, 2013). Although, no carcinogenic effects are
observed in chronic feedings of 100 mg/kg in a mouse, we did not want to risk
the integrity of the experiment. Therefore, only chronic doses of DCS were given
with a bolus of PIO (100 mg/kg) on the 7th day.
Control mice with TIC received an i.p. injection of normal saline (200
µl/mouse). Mechanical allodynia was evaluated at 0, 0.5, 1, 2, 3, 4, 5, and 6 hour
post injection time points.
4.2.4.2.3. Experiment 3: 7-day Treatment of 80 mg/kg Dose of DCS
Followed by a Bolus of 100 mg/kg Dose of PIO on the 7th Day
At least 8 weeks after the TIC injury, mice started receiving daily
subcutaneous injections of 80 mg/kg dose of DCS. On the 7th day, in addition to
the 80 mg/kg DCS dose, the mice were given a one-time bolus of PIO, 100
mg/kg (i. p.). In parallel to the mice with TIC injury, sham mice were also given
the same injections. The vehicle control mice with TIC injury and sham mice
received a daily subcutaneous injection of normal saline (150 µl). Mechanical
91
threshold was measured daily at 2.5-3 hours after injection. On the 7th day, the
mice with TIC injury (saline vs. drug group) were placed in the light/dark box for
preference testing.
4.2.4.2.4. Experiment 4: Single Dose of Mitochondrial Uncoupler
2,4-DNP
At least 8 weeks after TIC injury, the BALB/C mice were given either 5
mg/kg i.p. dose of 2,4, DNP or an i.p. injection of 10% DMSO (250 µl) as vehicle
control. Mechanical allodynia was assessed at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8
hour time point after injection.
4.2.5. Mitochondrial Isolation Assays
Isolated Mitochondrial assays were taken from previously described methods
(Pandya et al 2013; Pandya et al 2007; Sauerbeck et al 2011a; Sauerbeck et al
2012; Sauerbeck et al 2011b).
4.2.5.1.
Isolated Mitochondria Preparation
Twenty-eight weeks post TIC injury; mice were euthanized with CO 2 and
rapidly decapitated. Naïve age matched mice were also euthanized as controls.
The brainstems and brain were quickly dissected and placed on an ice cold
dissecting plate where the entire cortex was divided from the rest of the brain.
Then the entire cortex and whole brainstem were utilized for this experiment in
92
order to obtain enough tissue and mitochondria to perform the Seahorse assay.
The cortex and brainstem were homogenized in separate vials each containing 2
ml of 4°C mitochondrial isolation buffer (MIB) containing (215 mM mannitol,
75mM sucrose, 0.1% BSA, 20mM HEPES, 1mM EGTA, pH adjusted to 7.2 using
KOH). The EGTA (ethylene glycol tetraacetic acid), a calcium chelator, was
added to improve the mitochondria isolation (Pandya et al 2013; Patel et al
2009). Tissue homogenates were centrifuged twice at 1300 x G for 3 minutes at
4°C. The resultant supernatant was then removed and centrifuged at 13,000 x G
for 10 minutes at 4°C. The mitochondrial/synaptosomal pellets were burst in a
nitrogen bomb chamber (1200 psi for 10 minutes at 4°C). After the nitrogen burst,
the mitochondrial pellets were placed atop of a discontinuous Ficoll gradient
(7.5% - 10%) and centrifuged at 100,000 X G for 30 min at 4°C. The
mitochondrial pellet was then resuspended in EGTA-free MIB at 10,000 x G for
10 minutes at 4°C. The final pellet was resuspended at a concentration of 10
mg/ml in EGTA-free MIB and was stored on ice until further use. A BCA protein
assay kit determined the protein concentrations in a Biotek Synergy HT plate
reader by measuring absorbance at the optical wave length of 560nm (Winooski,
Vermont).
4.2.5.2.
Bioscience Seahorse XFe24 Flux Analyzer Assay
The Seahorse XFe24 Flux Analyzer (Seahorse Bioscience, Massachusetts,
United States) was used to measure the mitochondrial bioenergetics in isolated
mitochondria preparation as previously described (Pandya et al 2013; Sauerbeck
93
et al 2011b). The day before the experiment, the stock mitochondrial substrates
and inhibitors were prepared (500 mM pyruvate, 250 mM malate, 30 mM ADP, 1
mg/ml oligomycin-A, 1 mM FCCP, 1 mM rotenone, and 1 M succinate with the
pH adjusted to 7.2). The XF Calibrant solution (1ml) was added to each well of a
24-well calibration sensor cartridge. This sensor cartridge was then positioned
on the 24-well calibration plate and placed in a 37°C incubator overnight, the
calibration sensor cartridge ports A to D were loaded with the appropriate
mitochondrial substrates/inhibitors at 10x concentrations at the following day.
The mitochondrial respiration buffer (MRB) consisted of 215 mM mannitol, 75
mM sucrose, 0.1% BSA, 20 mM HEPES, 2 mM MgCl, 2.5 mM KH2PO4 adjusted
to a pH of 7.2. The volume of MRB in the mitochondrial plate was based upon
the original 500µl MRB volume.
The brain mitochondrial samples (10 µg) of both mice with TIC and naïve
mice were analyzed together on a single plate. After being resuspended in MRB,
50 µl of the mitochondrial samples were added in each experimental well with
control wells (totaling 4 wells) only obtaining 50 µl of MRB. The XF24 plate was
centrifuged at room temperature for 4 minutes at 3,000 rpm. For half of the TIC
and naïve mitochondrial samples (totaling 10 wells), 475 µl of MRB at 37°C was
added to each well. For the other half of the TIC and naive mitochondrial
samples (totaling 10 wells) 475 µl of MRB (37°C) containing 50 nM of DCS and
50 nM of PIO were added to each making a total volume in each well equal to
525 µl. The plates were then placed in the Seahorse XFe24 flux analyzer for
mitochondrial bioenergetics analysis following the calibration.
94
Sauerback and colleagues (Sauerbeck et al 2011b) provide a detailed
explanation of the cyclic protocol in which the appropriate substrates/inhibitors
are added to the wells and the measurement of the oxygen consumption rate is
recorded for each well. The substrates/inhibitors listed above were added from
port A to port D. Pandya and colleagues (2014) describes the State III response
after 5mM pyruvate, 2.5 mM malate, and 1 mM ADP were measured (Port A).
State III is a good indicator of how healthy the mitochondria providing a complex I
driven ADP phosphorylation rate and ATP synthesis. State IV response is in the
presence of all the State III substrates/inhibitors including the addition of 1 µM
oligomycin A (Port B). With the oligomycin A addition, State IV is inhibiting the
complex V (ATP synthase) action. The Seahorse analyzer will collect data and
report as percent Oxygen Consumption Ratio (OCR) at each State. Therefore,
State III/State IV is known as the Respiratory Control Ratio (RCR) which is used
to determine how well coupled electron transport is to the production of ATP. A
RCR that is greater than 5 is reported for healthy mitochondria.
4.2.6. Statistical Analysis
GraphPad Prism 6 software package (GraphPad Software, Inc. La Jolla, CA) was
used for graphing and statistical analysis of data from all behavioral tests, drug
administrations, and mitochondrial assays. Data is shown as mean ± standard
error of the mean (S.E.M.). Data was analyzed by two-way analysis of variance
(ANOVA) followed by Bonferroni post hoc test, or one-way ANOVA followed by a
95
Fishers post hoc test, or by standard, two-tailed, unpaired t-test (where is
appropriate). A p≤0.05 was considered a significant for all tests.
4.3.
Results
4.3.1. Unilateral Facial Mechanical Allodynia in Mice with TIC Injury
Baseline 50% mechanical thresholds were taken before surgery and were
similar for mice with TIC injury and sham (TIC: 3.84 ± 0.35 g vs. sham: 3.47 ±
0.00 g). However, one week post TIC injury the 50% mechanical threshold on the
ipsilateral whisker pad of the mice decreased to 0.37 ± 0.39 g and this was
significantly different from that on the contralateral side whisker pad (3.45 ± 0.04
g vs. 0.37±0.39 g; p<0.0001, two-way ANOVA, Bonferroni post hoc test; Figure
4.1). The unilateral lowered mechanical threshold started within the first week,
reached the maximum mechanical allodynia (threshold at the 0 g) at week 1 and
lasted until the animals were euthanized. The sham mice did not display any
differences in the mechanical threshold of the whisker pad before and postsurgery. The 50% mechanical threshold of the ipsilateral whisker pad of sham
mice was 3.41 ± 0.02 g and contralateral whisker pad was 3.43 ± 0.05 g.
Comparing the mechanical threshold of the ipsilateral side whisker pad between
the mice with/without TIC injury, there is also a significant difference (p<0.0001,
two-way ANOVA, Bonferroni post hoc test; n=9).
96
4.3.2. D-cycloserine Attenuated Mechanical Allodynia But Did Not Relieve
Anxiety Behaviors Associated with Hypersensitivity
Single doses of DCS (40 mg/kg, 60 mg/kg, 80 mg/kg, 100 mg/kg, 160
mg/kg, and 320 mg/kg) were given in ascending sequence with a one week
interval between each treatment, given to the mice with TIC injury to determine a
DCS dose response curve (Figure 4.2A). Only the higher DCS doses, 160 mg/kg
and 320 mg/kg, were effective in alleviating mechanical allodynia on the whisker
pad of the mice with TIC injury. At 3 - 4 hour post injection, the mechanical
threshold of the 160 mg/kg dose treatment group was elevated (hr 3: 0.51± 0.13
g; hr 4: 0.43 ± 0.12 g) and was statistically significant different from that of saline
treatment group. (hr 3: 0.00 ± 0.00 g; hr 4:0.01 ± 0.00 g; p<0.0001, two-way
ANOVA, Bonferroni post hoc test). The mechanical threshold of the 320 mg/kg
dose group was elevated at 3 hour post injection (0.17 ± 0.11 g) and was
statistically significant different from that of the saline control mice group (0.00 ±
0.00; p<0.0001, two-way ANOVA, Bonferroni post hoc test; n=4-8).
As shown in Figure 4.2B, the daily dose of 80 mg/kg attenuated the
mechanical allodynia in ipsilateral whisker pad of the mice with TIC injury on the
6th day of injection (1.10 ± 0.62 g; n=4-7). Interestingly, the 40 mg/kg and 60
mg/kg dose of DCS had no effect on the mice with TIC injury, but the sham mice
that received the 40 mg/kg dose of DCS became hypersensitive to the von Frey
fiber stimuli on the whisker pad bilaterally (0.37 ± 0.36 g; indicated by the orange
line in Figure 4.2B). The decreased 50% mechanical threshold was significantly
different from that of the shams that received the 60 mg/kg dose (3.76 ± 0.23 g)
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and the 80 mg/kg dose treatment (3.43 ± 0.05 g; p<0.001; two-way ANOVA,
Bonferroni post hoc test; n=4 - 7).
Previous studies have reported DCS is involved in alleviating anxietyrelated disorders (Antal & Paulus 2011; Davis et al 2006; Norberg et al 2008).
The light-dark box preference test was used to determine if the 7-day treatment
of DCS would attenuate the anxiety-like behaviors that developed in the mice
with TIC injury. Figure 4.2C depicts the time of light side occupancy of the mice
with TIC injury injected with saline (235.30 ± 55.80 sec) vs. the mice with TIC +
DCS (271.30 ± 65.16 sec; p=0.69; unpaired t test; n=5). Although there is no
statistical significant difference in the DCS treated mice compared to the saline
treated, the DCS treated mice did spend substantially more time in the light side.
This indicated that DCS had minimal effect at the dose tested and would be more
effective in relieving the anxiety-like behaviors in a higher doses range.
4.3.3. Attenuation of Mechanical Alloydnia and Anxiety by Combination of
Ineffective Low Doses of D-cycloserine and Pioglitazone
A single dose of 100 mg/kg of PIO (i.p.) was determined to be ineffective,
but the 300 mg/kg dose of PIO was effective in alleviating mechanical allodynia
in mice with TIC injury (see chapter 3). There are no negative interactions
reported between PIO and DCS in the literature. There is also no mention of the
effect of the two drug combination on orofacial neuropathic pain as was explored
with our TIC injury mouse model. Figure 4.3A depicts the one-time drug dose
combination of 100 mg/kg (i.p.) of PIO and 80 mg/kg (s.c.) of DCS compared to
98
these same doses given alone in the mice with TIC injury. The mechanical
allodynia was attenuated in the mice with TIC injury treated with the drug
combination of PIO +DCS (the 50% mechanical threshold was 0.94 ± .0.34 g)
and was significantly different compared to that in the saline treated animals
(0.00 ± 0.00 g; at hour 3 post injection p<0.001, two-way ANOVA, Bonferroni
post hoc; n=5-9; Figure 4.3B). The effect peak at 2 hour and lasted for 4 hours
post injection. The effect of the drug combination was also significantly different
from that of a single dose of 100 mg/kg PIO (0.03 ±0.01 g) or a single dose of 80
mg/kg DCS (0.06 ± 0.02 g). These one-time doses given alone had no effect on
mechanical allodynia in the mouse with TIC injury.
Following the same experimental method as above for 7-day dosing with
80 mg/kg (s.c.) of DCS, the study was repeated with the addition of a 100 mg/kg
(i.p.) bolus of PIO given on the 7th day. The drug combination on the 7th day had
a greater attenuating effect on mechanical allodynia compared to the effect in
saline treated mice. Two hours following the injection, the mice were placed in
the light-dark box for the preference test. The mice treated with the drug
combination (DCS + PIO) spent a significantly increased amount of time in the
light side compared to saline treated mice (drug: 221.0 ± 52.33 sec vs saline:
75.28 ± 36.65 sec; p<0.05, unpaired t-test; Figure 4.3C). Taken together, these
data demonstrated that combination of PIO and DCS at low dose has a
potentiating effect. Additionally, this drug combination provided a reversal of
anxiety-like behaviors associated with the TIC injury, suggesting it could be very
99
beneficial in clinic patients who suffer from chronic orofacial pain and anxietyassociated with pain.
4.3.4 2.4-DNP Attenuated Mechanical Allodynia in Mice with TIC Injury
The mitochondrial uncoupler, 2,4-DNP, has been proven to be
neuroprotective after traumatic brain injury (Pandya, 2007). At week 8 post injury,
5 mg/kg of 2,4-DNP, single dose injection (i.p.) effectively attenuated mechanical
allodynia on the whisker pad of the mice with TIC injury (Figure 4.4). The effect
started from 2 hour post injection (1.03 ± 0.15 g) and lasted for 5 hour (3hr: 1.64
± 0.21g; 4hr: 1.17 ± 0.09g: 5hr: 0.83 ± 0.07g). This difference was statistically
significant compared to mice with TIC injury given vehicle (10% DMSO) (0.02 ±
0.00g; *p<0.05, ****p<0.0001; two-way ANOVA, Bonferroni post hoc test). The
effectiveness of this drug in increasing mechanical threshold suggests that
mitochondrial dysfunction could be a key factor in maintaining the chronic
mechanical allodynia in the mice after TIC injury. Thereby, drugs that uncouple
the altered mitochondrial function could be beneficial for relieving chronic pain.
4.3.5. Cortex Mitochondria Had a Decreased Respiratory Control Ratio in Mice
with TIC Indicating Cortical Mitochondrial Dysfunction
The mitochondrial isolation assay and Seahorse XFe24 analyzer was used
to determine the mitochondrial oxygen consumption of isolated cortical
mitochondria in the mice with TIC injury. The State III OCR of the mitochondria
from mice with TIC injury (382.40 ± 10.47) significantly increased compared to
the State III OCR of the mitochondria from naïve animals (253.9 ± 11.99;
100
p<0.001, two-way ANOVA; Figure 4.5A). The oxygen consumption rate (OCR) of
State IV increases in the mitochondria from mice with TIC injury compared to the
mitochondria from the naïve controls (TIC: 86.78 ± 1.93 vs. 47.14 ± 8.54; p<.05,
two-way ANOVA; Figure 4.5B). This data suggest that the cortical mitochondria
from mice with TIC injury had increased mitochondrial bioenergetics. However,
the Respiratory Control Ratio (RCR), defined as RCR = State III/State IV,
significantly decreased in the mitochondria from mice with TIC injury compared to
the RCR in the mitochondria of naïve controls (TIC: 4.41 ± 0.07 vs. naïve: 6.64 ±
1.01; p<0.05, two-way ANOVA; Figure 4.5C).
These results indicate
mitochondrial dysfunction occurs in the mice with TIC injury since the RCR is
less than 5.
The DCS/PIO drug combination (added ex vivo to the MRB) did not
significantly change State III or State IV OCR. However, the drug combination did
significantly increase the RCR of the cortical mitochondria of mice with TIC
(cortex: 6.00 ± 0.71) compared to untreated mitochondria of mice with TIC
(cortex: 4.407 ± 0.07, p<0.5, unpaired t-test; Figure 4.5C). The drug combination
significantly decreased the RCR of the cortical mitochondria of naïve mice (4.08
± 0.21) compared to that of untreated cortical mitochondria from naïve controls
(6.64 ± 1.008; p<0.5, two-way ANOVA; Figure 4.5C).
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4.3.6. Drug Combination Treatment Increased Respiratory Control Ratio in
Brainstem Mitochondria of Mice with TIC Injury
Isolated brainstem mitochondria of the mice with TIC injury were analyzed along
with the mitochondria from age matched naïve mice. The OCR of State III and
State IV of the mitochondria from mice with TIC injury (state III: 502.10 ± 48.26;
state IV: 70.44 ± 9.43) was not significant compared to the mitochondria of
naïves (state III: 513.90 ±27.81; state IV: 85.87 ± 9.17; Figure 4.6A & 6B). The
RCR of the mitochondria of the mice with TIC injury (7.43 ±0.34) compared to the
mitochondria from naïve mice was not significantly different (6.24 ± 0.35; Figure
4.6C). However, with the two drug combination, the only significant change
observed was that the mitochondria drug treated mice with TIC injury had an
increased RCR compared to the mitochondria from the untreated mice with TIC
injury (treated: 9.00 ± 0.59 vs. untreated: 7.43 ± 0.34, p<0.05, two-way ANOVA,
Figure 4.6C).
4.4. Discussion
In this study, it was determined that higher doses of single injections of
DCS (160 mg/kg and 320 mg/kg) were efficacious in alleviating mechanical
allodynia on the whisker pad of mice with TIC injury. However, low doses of DCS
such as 40 mg/kg induced hypersensitivity in the sham operation control animals.
Chronic treatment with the 80 mg/kg dose of DCS for 7 days partially attenuated
mechanical allodynia on the whisker pads of the mice with TIC injury on the 6 th
day. This chronic treatment dose, however, did not improve the anxiety-like
102
behavior associated with TIC injury as shown by the light-dark box preference
test. The combination of the low dose of DCS (80 mg/kg) and PIO (100 mg/kg)
was shown to significantly increased the 50% mechanical threshold levels of the
mice with TIC injury compared to that in saline treated mice with TIC injury as
well as that in the ineffective single dose of 100 mg/kg PIO or 80 mg/kg DCS.
Furthermore, the 7-day dose regiment of 80 mg/kg dose of DCS was repeated
with the exception of the additional bolus of 100 mg/kg dose of PIO given on the
7th day. This was to mimic the DCS (single drug only) 7-day regiment followed by
the light-dark box preference test. Single dose of drug combination treatment
improved not only mechanical allodynia, but also the anxiety-like behavior
associated with the TIC injury.
In this study, the mild mitochondrial uncoupler, 2,4-DNP, significantly
elevated the 50% mechanical threshold on the whisker pads of the mice with TIC
injury. Although this provides evidence that mitochondrial dysfunction occurs in
the mice with TIC injury, ex vivo studies were conducted in order: 1) to confirm
the mitochondrial dysfunction after the TIC injury and 2) to determine if the drug
combination (DCS\PIO) is acting specifically on the mitochondria to attenuate
mechanical allodynia. The mitochondrial isolation assays determined that State
III and State IV OCR increased in cortical mitochondrial, but did not change in
brainstem mitochondria. However, the RCR did in fact decrease in the isolated
cortical mitochondria. Furthermore, when treated with the drug combination, the
RCR of the cortex mitochondria in the naïve mice decreased while those of the
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mice with TIC injury increased not only in the cortex mitochondria but also in the
brainstem mitochondria.
Although clinical trials have been conducted with DCS for alleviation of
chronic back pain, anxiety/stress disorders, and fear related to pain (Davis et al
2006; Heaton et al 2010; Norberg et al 2008), only one study has been published
that observes its attenuation of chronic orofacial pain (Antal & Paulus 2011).
DCS is known to have an affinity for a specific glycine binding site on the NMDA
receptor. Previous studies that have shown that at lower doses, DCS will act as a
partial agonist on the NMDA receptor producing hypersensitivity, but higher
doses, DCS can act as a partial antagonist of NMDA (Kushner et al 2007;
Lanthorn 1994). Similarly, the present study found that DCS acts as an agonist at
low doses (40 mg/kg) inducing hypersensitivity in naïve animals and as an
antagonist at high doses (160, 320 mg/kg) in the mice with TIC injury for
alleviation of chronic pain. As explanation for the dual effect, Mony and
colleagues speculated that many ligands in high doses desensitizes the NMDA
receptor (Mony et al 2009).
Although previous studies have determined that PIO, a PPARγ receptor
agonist, attenuates neuropathic pain and inflammatory nociception due to
peripheral nerve injury (Fehrenbacher et al 2009; Ghosh et al 2007; Maeda &
Kishioka 2009; Morgenweck et al 2010; Morgenweck et al 2013; Park et al 2007),
the present study was the first to use the drug combination of DCS and PIO to
treat orofacial neuropathic pain and anxiety associated with TIC injury in a mouse
model. It was determined that using ineffective low doses of both DCS and PIO,
104
when combined together, attenuated trigeminal neuropathic pain in the mice with
TIC injury. This potentiated effect also improved the anxiety-like behavior in the
mice with TIC injury whereas the DCS only treatment did not. This indicated that
the drug combination is having a potentiated effect most likely in higher order
brain regions to attenuate not just pain, but anxiety-related to pain (Dellarole et al
2014; Lipton et al 2000; McWilliams et al 2003; McWilliams et al 2004; Robinson
et al 1988). Furthermore, the DCS/PIO combination produced no overt side
effects observable in the mice with TIC injury. Therefore this drug combination
could be a very beneficial treatment for patients who are suffering from
depression, anxiety, or other psychological conditions due to their chronic pain
status.
Although DCS and PIO could be acting through the NMDA receptor and
PPARγ separately, another explanation for this potentiated effect is that these
drugs are acting through alternative receptors altogether. In fact, some studies
have supported a role for PIO acting through PPARγ independent pathways
directly on mitoNEET, a mitochondrial protein. mitoNEET is vital for mitochondrial
respiration and for increasing mitochondrial bioenergetics (Geldenhuys et al
2014; Wiley et al 2007a; Wiley et al 2007b; Yonutas & Sullivan 2013).
Furthermore, Korde and Maragos (2012) successfully identified a NMDA-like
receptor directly on the mitochondrial membrane (Korde & Maragos 2012). This
evokes the questions: Is the DCS/PIO combination alleviating trigeminal pain by
correcting mitochondrial dysfunction through these mechanisms?
105
Although mitochondrial dysfunction has been shown to be responsible for
maintaining chronic pain after neuronal injury (Bouillot et al 2002; Ferrari &
Levine 2010; Joseph & Levine 2006; Kim et al 2004; Shin et al 2003; Sui et al
2013), this is the first study to demonstrate that mitochondrial dysfunction occurs
in a chronic trigeminal neuropathic pain model. First, this study determined that
a mild mitochondrial uncoupler, 2,4-DNP, attenuated mechanical allodynia on the
whisker pad of the mice with TIC injury. This was supportive reasoning that
mitochondrial dysfunction was occurring in the mice with TIC. However, it did not
answer the question of whether mitochondrial function could be improved with
the DCS and PIO drug combination.
To further investigate the role of mitochondria in neuropathic pain after
TIC injury, isolated mitochondrial assays were performed at 28 weeks post injury.
This study observed the mitochondrial respiration rates of States III and IV in
particular. These data showed that the cortical mitochondrial of the mice with TIC
injury have increased State III respiration as well as an increased State IV
respiration, thereby significantly decreasing the RCR compared to naïve mice.
However, there were no significant changes in the brainstem mitochondria
compared to naive mice. This data could be interpreted in many ways.
First, this data could support an adaptive mechanism that is occurring in
the cortical mitochondrial of the injured animals. Since the mitochondria was
analyzed 28 weeks post injury, it is sufficient to say that in a chronic state the
increased State III respiration is needed to provide sufficient ATP production for
brain function. However, while State III indicates complex I driven ADP
106
phosphorylation and the general mitochondrial oxidation, State IV is a sufficient
indicator of electron leak (Brand & Nicholls 2011; Chance & Williams 1955a; b).
Since State III respiration also increased, this could be supportive of increased
electron leak that then leads to increased ROS production. However, further
studies need to be conducted to confirm mitochondrial ROS in the mice with TIC
injury. Since the respiratory control ratio (RCR) (State III/State IV) was less than
5 in the cortical mitochondrial of the mice with TIC injury, this would support the
idea that the complexes of electron transport chain are not very well coupled to
net production of ATP, indicating mitochondrial dysfunction (Brand 1990; Brand
et al 1978; Brand & Nicholls 2011).
Furthermore, the drug combination (DCS/PIO) increased RCR in the
cortical and brainstem mitochondria, but decreased RCR in the cortical
mitochondria of the naïve mice. The DCS/PIO combination was able to improve
RCR suggesting that the DCS/PIO combination improves mitochondrial
dysfunction after injury, but is not beneficial in naïve mice. An underlying detailed
mechanism for the DCS and PIO combination effect is unknown and needs to be
investigated in future studies. Future studies could confirm if this drug
combination is acting through protein-dependent (NMDA-like receptor in
mitochondria for DCS and mitoNEET for PIO) or protein-independent
mechanisms to improve mitochondrial bioenergetics. Future studies looking at
specific complex activity of the mETC, in particular the redox state of complex I
could be beneficial in uncovering the mechanism. (Starkov 2006).
107
Additionally, mitochondrial dysfunction was only detected in the cortical
mitochondria, but not in the brainstem. This could be due to the fact that there is
a large area of cortex dedicated to the trigeminal somatotopic map, and thus, a
higher number of mitochondria in the entire cortex affected compared to the
brainstem. The trigeminal dorsal horn, on the other hand, is a small portion of the
brainstem. Nevertheless, the drug combination was acting to increase RCR at
both levels to ADP phosphorylation. This paralleled improvement of both aspects
of pain demonstrated with the behavioral tests.
Lastly, unlike 2,4-DNP which was removed from the market due to
increased fatality rates, DCS and PIO, given at their low ineffective dose
combination, did not induce any observed side effects in the mice. The DCS\PIO
combination could be a great value in the clinic with patients suffering from
continuous trigeminal neuropathic pain.
4.5.
Conclusion
This study demonstrated that the combination of DCS/PIO attenuated
not only orofacial neuropathic pain, but also the anxiety behaviors associated
with the TIC injury through receptor independent mechanisms. These
mechanisms were supported by the DCS/PIO combination improving the cortical
mitochondrial dysfunction present in the mice with TIC injury.
108
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
5
S h a m Ip s ila te r a l
4
S h a m C o n t r a la te r a l
T IC Ip s ila t e r a l
3
T IC C o n t r a la t e r a l
2
1
****
0
2
-1
4
6
8
10
T im e ( w e e k s )
Figure 4.1. TIC Injury Induced Unilateral Whisker Pad Mechanical Allodynia.
The 50% mechanical threshold (in gram force) was measured bilaterally on the
whisker pads of the mice with TIC injury and the sham mice. The mechanical
threshold was decreased on the ipsilateral whisker pad of mice with TIC injury
within one week post injury. The mechanical threshold of contralateral whisker
pad was unaffected by surgery. The mechanical threshold of the ipsilateral and
contralateral whisker pads of the sham mice also did not change. TIC n=9; TIC
(ipsi) vs. TIC (con.) or naïve ****p<0.0001, two-way ANOVA, Bonferroni post hoc
test.
109
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
A
4 .0
3 .5
C o n tr o l T h r e s h o ld
3 .0
T IC S a lin e
2 .5
T IC 4 0 m g /k g D C S
2 .0
T IC 6 0 m g /k g D C S
1 .5
T IC 8 0 m g /k g D C S
****
****
1 .0
0 .5
T IC 1 0 0 m g /k g D C S
****
*
T IC 1 6 0 m g /k g D C S
T IC 3 2 0 m g /k g D C S
0 .0
0
1
2
3
4
5
T im e ( h )
B
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
N A IV E
N A IV E 8 0 m g /k g
6
S H A M S a lin e
5
S H A M 4 0 m g /k g D C S
S H A M 6 0 m g /k g
4
S H A M 8 0 m g /k g
3
T IC S a lin e
T IC 4 0 m g /k g D C S
***
2
T IC - 6 0 m g /k g
1
T IC 8 0 m g /k g D C S
0
0
1
2
3
4
5
6
7
8
T im e ( d a y s )
C
D u r a t io n in L ig h t ( s e c )
400
T IC : S a lin e
T IC : D C S
300
200
100
0
Figure 4.2. DCS Attenuated Mechanical Allodynia, but Does Not Reverse
Anxiety-Like Behavior in the Mice with TIC Injury. (A) Dose response curve
for DCS showed that higher doses of DCS (160 mg/kg and 320 mg/kg) are
110
effective at alleviating the mechanical allodynia on the whisker pad of the mice
with TIC injury. (n=6) (B) The mice were given a 7-day treatment of lower doses
of DCS (s.c.). Only the 80 mg/kg dose elevated the mechanical threshold in the
mice with TIC injury. The 40 mg/kg dose of DCS lowed the mechanical threshold
on the whisker pad of the sham mice while the 60 mg/kg dose had no effect on
sham mice or mice with TIC injury (n=8). (C) On day 7, two hours post-injection,
the mice with TIC injury treated with 80 mg/kg dose of DCS or went through the
light-dark box preference test. There is no difference in the time of the light side
occupancy between drug treated and vehicle treated group. *p<0.05, ***o,0.01;
****p<0.0001; two-way ANOVA, Bonferroni post hoc test. Figure C: Unpaired ttest, n.s.; n=5.
111
A
5 0 % m e c h a n ic a l th r e s h o ld
4 .0
3 .5
C o n t r o l T h r e s h o ld
3 .0
S a lin e
2 .5
DCS
2 .0
P IO
1 .5
P IO + D C S
***
**
1 .0
*
0 .5
0 .0
0
1
2
3
4
5
6
7
T im e ( h )
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
B
4 .0
S h a m (s a lin e )
3 .5
S h a m ( D C S + P IO )
3 .0
T IC ( s a lin e )
2 .5
T IC ( D C S + P I O )
2 .0
****
1 .5
1 .0
0 .5
0 .0
0
1
2
3
4
5
6
7
8
T im e ( d a y s )
C
D u r a tio n in L ig h t ( s e c )
300
*
250
T IC : S a lin e
T IC : D C S + P I O
200
150
100
50
0
112
Figure 4.3. The Combination of DCS and PIO Attenuated Mechanical
Allodynia and Anxiety-Like Behavior in the Mice with TIC Injury. (A) Onetime dose of DCS (80 mg/kg) + PIO (100 mg/kg) provided a potentiated effect in
elevating mechanical threshold of the whisker pad of the mice with TIC injury
which was significantly different than that of the single dose of either DCS (80
mg/kg) only or PIO (100 mg/kg) only (n=6-8). The mechanical threshold of the
drug combination treatment group was also significantly different from the vehicle
treated group. (B) After a 7-day dose of DCS (80 mg/kg), a bolus of PIO (100
mg/kg) was give on the 7th day. The mechanical threshold was dramatically
increased in the mice with TIC injury. This was significantly different from that of
the vehicle treated mice with TIC injury. (C) Two hours following injection of the
PIO bolus on day 7, the drug and the vehicle treated mice with TIC injury went
through in the light-dark box preference test (n=8/group). The drug treated group
showed a significant increase in the time spent in the light chamber compared to
that of the vehicle treated group (*p<0.05, ***p<0.001; ****p<0.0001; two-way
ANOVA, Bonferroni post hoc test. Figure C: Unpaired t-test, (*p<0.05.; n=5).
113
5 0 % M e c h a n ic a l T h r e s h o ld ( g )
4 .0
3 .5
C o n t r o l T h r e s h o ld
3 .0
T IC : V e h ic le ( 1 0 % D M S O )
2 .5
T IC : 2 ,4 D N P (5 m g /k g )
2 .0
****
1 .5
****
****
1 .0
****
*
0 .5
0 .0
0
1
2
3
4
5
6
7
T im e ( h )
Figure 4.4. A Mild Mitochondrial Uncoupler Attenuated the Mechanical
Allodynia in the Mice with TIC Injury. 2,4-DNP (5 mg/kg) significantly
increased the 50% mechanical threshold of the mice with TIC injury compared to
that of the vehicle treated mice. This effect started at one hour, peaked at 3 hour
and lasted for 5 hours post injection. (n=8; (*p<0.05, ****p<0.0001; two-way
ANOVA, Bonferroni post hoc test.).
114
*
500
N a iv e
400
(p M o l/m in )
O x y g e n C o n s u m p tio n R a te
S ta te III:
A
N a iv e : D C S + P I O
300
T IC
T IC : D C S + P I O
200
100
0
*
B
*
N a iv e
N a iv e : D C S + P I O
80
(p M o l/m in )
S ta te IV :
O x y g e n C o n s u m p tio n R a te
100
T IC
60
T IC : D C S + P I O
40
20
0
**
C
10
*
R C R fo r C o rte x
N a iv e
N a iv e : D C S + P I O
8
#
6
T IC
T IC : D C S + P I O
4
2
0
Figure 4.5. Isolated Cortical Mitochondria from the Mice with TIC Injury
Showed an Increased State III and State IV OCR, but Decreased RCR which
is Reversed With the Drug Combination of DCS + PIO. Cortical mitochondria
isolated from mice with TIC injury had an increased oxygen consumption rate
(OCR) for State III (A) and increased OCR in State IV respiration (B). This is
115
significantly different compared to those of the cortical mitochondria from naïve
mice. The respiratory control ratio (RCR = StateIII/StateIV)) decreased in the
cortical mitochondria of the mice with TIC injury (C). However, the drug
combination of DCS and PIO decreased the OCR in State IV, therefore
increased the RCR (n=8/group; *p<0.05, **p<0.01; two-way ANOVA, Bonferroni
post hoc test; #p<0.05, unpaired t-test).
116
A
N a iv e
*
N a iv e : D C S + P I O
600
(p M o l/m in )
S ta te III:
O x y g e n C o n s u m p tio n R a te
800
T IC
T IC : D C S + P IO
400
200
0
B
N a iv e
N a iv e : D C S + P I O
(p M o l/m in )
S ta te IV :
O x y g e n C o n s u m p tio n R a te
150
T IC
100
T IC : D C S + P I O
50
0
C
15
R C R f o r B r a in s t e m
N a iv e
N a iv e : D C S + P I O
10
*
T IC
T IC : D C S + P IO
5
0
Figure 4.6. The Drug Combination (DCS +PIO) Increased the Brainstem
Mitochondrial RCR in the Mice With TIC Injury. There are no significant
differences in the OCR of State III (A) or State IV (B) for the isolated brainstem
mitochondria from the mice with TIC injury compared to that of the naïve mice.
The respiratory control ratio (RCR) did not significant change in the isolated
117
brainstem mitochondrial from the mice with TIC injury compared to that of naïve
mice (C) However, due to the drug combination treatment (50 nM DCS+ 50nM
PIO, ex vivo) increased the OCR of the brainstem mitochondria of mice with TIC
injury at the state III as showed in (A). The RCR of the brainstem mitochondria
from the mice with TIC injury did increase (n=4/group; *p<0.05; two- way
ANOVA, Bonferroni post hoc test).
118
CHAPTER FIVE
OVERALL DISCUSSION AND CONCLUSIONS
The aims of the study were to develop a new orofacial neuropathic pain
model specifically for mice that provides a suitable model for study of orofacial
pain. With this model, the studies sought to identify certain molecular targets that
play a major role in the etiology and maintenance of trigeminal neuropathic pain.
5.1. Trigeminal Inflammatory Compression (TIC) Injury Is a Clinically
Relevant Orofacial Neuropathic Pain Model
This series of studies expands our understanding of the novel facial
mouse pain model known as Trigeminal Inflammatory Compression (TIC) injury.
This chronic neuropathic pain model was described in our previous study (Ma et
al 2012a), but the cold, anxiety- and depressive-like behavioral characteristics of
this injury were not yet completely characterized. The TIC model demonstrated
several aspects consistent with chronic facial pain in humans. The first study of
this thesis determined that the surgery is 100% efficacious for producing facial
hypersensitivity in all of the animals receiving the chromic gut suture. The
placement of the suture provides mechanical allodynia with distinctive receptive
fields (on the affected whisker pad). Likewise, the pattern of receptive fields were
not always the same in all animals (depending on the nerve fascicle contacted by
the chromic gut suture), as the pattern of receptive fields from person to person
may also vary (Simons & Travell 1981; Travell 1981). The development of cold
119
allodynia on the whisker pad of the mice with TIC injury, but not heat
hypersensitivity is consistent with the clinical profile. Clinic patients suffering with
trigeminal neuropathic pain are not often bothered by heat stimuli, but instead
most often complain that light touch, and cold stimuli (De Leeuw 2008;
Zakrzewska 2013).
Furthermore, the studies determined that the chronicity of the TIC injury
model persists at least through 21 weeks. These were the first recorded data
indicating a mouse facial pain persisting through this long time course without
causing ischemia and complete demyelination of the infraorbital nerve (Ma et al
2012a). Accompanying the hypersensitization were the anxiety- and depressivelike behaviors. These were supported by the performance of the mice in operant
tests (light-dark box, open field, elevated plus maze). The anxiety-depressive-like
behavior was worsened by the acoustic disturbance.
This again fits into the definition of chronic pain described Wall & Melzack
(1999) with the two main features of “pain sensation” and “pain affect” which are
incorporated into the emotional distress suffered by patients with pain (Wall &
Melzack 1999). The “pain affect” has also been described as the sequelae of
other physical and psychological disorders such as anxiety and depression
(Dellarole et al 2014; Maletic & Raison 2009; McWilliams et al 2004). These
comorbidities have been so prevalent that approximately half of all patients
suffering from chronic pain reportedly also suffered from anxiety and depression
(Asmundson & Taylor 2009; Carleton et al 2009; Macianskyte et al 2011;
Robinson et al 2009). The relationship between chronic pain, depression,
120
anxiety, and fear is extremely complex with extensive overlap making it difficult to
determine a pattern of cause and effect. This complex relationship is related to
the numerous brain structures, such as the somatosensory cortex, prefrontal
cortex, nucleus accumbens, insular cortex, anterior cingulate cortex, amygdala,
hippocampus, thalamus, and cerebellum that have all been highlighted as major
players involved not only in pain perception but also in affective states, including
depression, anxiety, and fear (Apkarian 2004; Apkarian et al 2004a; Apkarian et
al 2004b; Becerra et al 2006; Dellarole et al 2014; Heim et al 2004; Robinson et
al 2009). Thus, the TIC injury model is indeed a representative model of anxietyrelated chronic facial neuropathic pain.
5.2. PPARγ Is an Important Molecular Target for the Treatment of
Trigeminal Neuropathic Pain
Our experiment determined that the nuclear receptor, peroxisome
proliferator receptor-gamma isoform (PPARγ), plays a significant role in
trigeminal pain transmission. Immunohistological study revealed that 3 weeks
after the Trigeminal Inflammatory Compression (TIC) injury, a most intense
PPARγ immunoreactivity appeared in the spinal trigeminal caudalis where the
primary nociceptive fibers synapse. Systemic administration of a PPARγ agonist,
pioglitazone (PIO), attenuated the mechanical allodynia in the mice with TIC
injury at doses of 300 mg/kg i.p. and 600 mg/kg p.o. However, 100 mg/kg of PIO
(i.p.) had no effect. Furthermore, these studies revealed that administering a
PPARγ antagonist, GW9662 (30 mg/kg i.p.) prior to the optimal dose of PIO (300
121
mg/kg i.p.) blocked the analgesic effect of PIO indicating that PIO is acting
through a PPARγ activation mechanism. Additionally, the PPARα agonists,
benzafibrate and fenofibrate, had no effect on the allodynic mice. However, the
PPARβ/δ agonist, GW0742 had a minimum alleviation of allodynia effect to the
mice with TIC, but it is not as effective as the PPARγ agonists. Taken together,
these results confirm PPAR’s role in trigeminal pain transmission, particularly
signaling through the γ isoform.
Furthermore, the combination of low doses of DCS (80 mg/kg) and PIO
(100 mg/kg) were shown to significantly elevate the 50% mechanical threshold of
the mice with TIC injury. The single dose of DCS only at 80 mg/kg or PIO at 100
mg/kg was shown to be ineffective. In combination, they produced a potentiated
effect. Treatment with the single dose combination improved not only mechanical
allodynia, but also the anxiety-like behavior associated with the TIC injury.
Therefore, the question is: What defines a molecule as a potential target
for the treatment of inflammation? Several features should be associated with the
molecular signature of an inflammatory target. First, the cellular or sub-cellular
expression of the molecule of interest could be changed upon inflammation.
Second, the activation of this molecule could cause signs of decreased
inflammation. Third, the functions of the molecule of interest could be regulated
by other inflammatory mediators. Finally, the inhibition or activation of this
molecule
should
downregulate
pro-inflammatory
mediators
that
cause
inflammation. After reviewing the data, this study demonstrated that PPAR-
122
gamma meets all the criteria, thereby supporting this receptor as a potential
therapeutic target to treat trigeminal neuro-inflammatory pain.
This study is not only the first study to identify PPARγ immunoreactivity
throughout the trigeminal brainstem sensory nuclear complex with/without
trigeminal nerve injury, but this study also is the first to demonstrate that PPARγ
activation attenuates trigeminal neuropathic/inflammatory pain. In conclusion,
PPARγ is suggested as a potential therapeutic target in the trigeminal pain
neuraxis. And from this study also arises the possibility of repurposing the FDA
approved diabetic therapeutic drug, PIO, for the treatment of patients suffering
from orofacial pain.
5.3. Mitochondrial Dysfunction is One Underlying Mechanism of Trigeminal
Neuropathic Pain
In this study, the mild mitochondrial uncoupler, 2,4-DNP, significantly
elevated the 50% mechanical threshold on the whisker pads of the mice with TIC
injury. This provides evidence that mitochondrial dysfunction occurs in the mice
with TIC injury. Ex vivo studies were conducted in order to determine: 1) the
cause of the mitochondrial dysfunction, and 2) whether the drug combination
(DCS\PIO) is acting on the mitochondria to attenuate mechanical allodynia. The
mitochondrial isolation assays determined that the OCR of State III and State IV
increased in cortical mitochondria, but did not change in brainstem mitochondria.
However, the RCR did in fact decrease in the isolated cortical mitochondria.
Furthermore, when treated with the DCS/PIO combination ex vivo, on the
123
isolated brain mitochondria preparation, the RCR of the cortex mitochondria in
the naïve mice decreased while those of the mice with TIC injury increased not
only in the cortex mitochondria but also in the brainstem mitochondria. Although
mitochondrial dysfunction has been shown to be responsible for maintaining
chronic pain after neuronal injury (Bouillot et al 2002; Ferrari & Levine 2010;
Joseph et al 2004; Joseph & Levine 2006; Kim et al 2004; Park et al 2006;
Schwartz et al 2009; Shin et al 2003; Sui et al 2013), this is the first study to
demonstrate that mitochondrial dysfunction occurs in chronic trigeminal
neuropathic pain. This study determined that a mild mitochondrial uncoupler, 2,4DNP, attenuated mechanical allodynia on the whisker pad of the mice with TIC
injury. The experiment suggests that the DCS/PIO combination could be acting in
the brain after TIC injury to improve the brain mitochondrial respiration capacity
and leads to highly efficient functional performance of the brain, thereby,
controlling pain related behavior and pain related anxiety.
5.4. Limitations and Future Studies
Several limitations exist for this study. First, all of our animals were male
C57Bl/6 mice. C57Bl/6 mice were chosen for their moderate levels of anxietylike behavior (Belzung 2001; Belzung & Griebel 2001; Kulesskaya et al 2014;
Kulesskaya & Voikar 2014). In order to collect more consistent results, females
or other strains were not tested in this study. However, future studies should be
conducted to observe the behaviors of different species. Since facial neuropathic
pain in humans is diagnosed more often in females,
124
behavioral studies
performed in chapter two need to be repeated in female mice with TIC injury
(Koopman et al 2009; Macfarlane et al 2002a; b; Macfarlane et al 2001; Rauhala
et al 2000).
Secondly, the operant behavioral tests were only conducted weeks 1,4,
and 8 post injury. This was done to prevent over-testing of the animals since
many previous studies suggest only testing occasionally (Walf & Frye 2007).
However, while anxiety- and depressive-like behaviors could have developed at
time points earlier than week 8 post injury that our experimental design would not
have detected, Yalcin (2007) reported that anxiety-like behavior starts to develop
at 6 weeks post injury. Therefore, a new experimental design could utilize
different time points such as 2, 6, 10 post injury to observe earlier and later time
points than 8 weeks post injury to help determine which week precisely the
anxiety-and depressive-like behavior starts to occur.
Thirdly, although depressive-like behavior was observed in mice with TIC
injury, we did not conduct any traditional operant depressive tests such as forced
swimming test, tail suspension test, and sucrose consumption test (Wang et al
2012; Zhang et al 2012a). Because we wanted to measure anxiety-like behavior
along with depressive-like behavior, assays such as the forced swimming and tail
suspension tests were not used because they subject the animals to an
extremely life threatening, stressful environment (Borsini & Meli 1988;
Sakakibara et al 2005). However, the sucrose consumption test would be an
ideal operant test to use to measure depression in the mice with TIC injury as
well as observe their reward-seeking behavior ((Wang et al 2012; Wyvell &
125
Berridge 2000). Since the mice with TIC revealed poor decision making ability in
the elevated plus maze test after exposure to the acoustic startle stimulus, the
sucrose test would also serve the purpose of determining the anhedonic behavior
of mice with TIC injury. Taken together, future studies combined with the data
collected here could provide better characterization of the cognitive impairment of
the mice with the TIC injury.
Another limitation of these studies is that all of the drug tests using PPAR
agonists/antagonists were one time dose administrations. In order to insure
efficacy in reducing mechanical allodynia in the mice with TIC injury, higher
doses were administered to the animals than preferable. The only overt side
effect that was observed was hypothermia when the animals were given the 300
mg/kg dose of PIO. Since toxic doses of PIO are report to be >181 mg/kg,
chronic feeding of mice at such high doses was not safe to perform. Therefore, it
would be beneficial in future studies to observe chronic treatment of PIO in the
mice with TIC injury at lower doses that have been previously published (Lee et
al 2006; Morgenweck et al 2013; Takamura et al 1999). Morgenweck and
colleagues (2013) pretreated their mice with PIO (10 mg/kg i.p. or 30 mg/kg p.o.
twice daily) before the spared nerve injury and observed partial inhibition of
mechanical allodynia (Morgenweck et al 2013). An additional experiment would
be to modify the treatments in this study for chronic feeding. Instead of
pretreating the mice with TIC with chronic low doses of PIO, treatment of PIO
would start at least 8 weeks post injury. This would be a beneficial way to test
PIO’s effects on mechanical allodynia in the mice with TIC after chronic
126
hypersensitivity and anxiety-like behaviors have already been established (see
chapter 2). Thereby, this study would answer the question as to whether PIO
could be translatable to the clinic to attenuate pain in patients suffering with
continuous trigeminal neuropathic pain.
Furthermore, previous studies have speculated that by providing chronic
treatment, PIO will act through PPARγ receptor signaling as a transcription factor
thereby downregulating pro-inflammatory cytokines rather than acting through
PPARγ receptor activated non-genomic pathways to reduce neurogenic
inflammation (Fehrenbacher et al 2009; Feinstein et al 2005; Gardner et al 2005).
Since PPARγ’s transcription actions takes longer than PPARγ’s rapid
nongenomic actions, this leads to strong speculation that low doses will be more
effective over a period of time rather acting rapidly as it did with the one-time
dose given in this study.
The histology done in this study was helpful not only to show localization
of PPARγ immunoreactivity in the TBSNC, but also to demonstrate that PPARγ is
upregulated three weeks after injury in the TBSNC. Future studies could observe
the alteration of immunoreactivity of PPARγ at different time points after TIC
injury such as 6, 9, 12 weeks post. This could be performed simultaneously with
a double-labeling of cytokines (IL-6, TNF-α) as well as glial markers (IB-4, OX42). This would provide insight into the relationship that is possibly occurring
between the neural immune system and the upregulation of PPARγ.
For the DCS/PIO combined treatment study, one limitation is that only one
drug dose combination was used (80 mg/kg DCS + 100 mg/kg PIO). Future
127
studies should be conducted at different dose combinations to determine if lower
doses of both drugs would elicit a similar or more effective anti-allodynic effect. In
this study, the mice with TIC were only given 7 days of DCS treatment and on the
7th day received a bolus of PIO. It would be more interesting to determine if
simultaneous chronic feeding of both DCS and PIO resulted in a greater increase
in mechanical threshold. As stated above, a future study would be to determine
an effective chronic treatment dose of PIO. Once determined, the low dose of
PIO and DCS could be given chronically together. Based on the data from the
study in which the one-time low dose combination of DCS/PIO attenuated
mechanical allodynia, speculation would lead to the theory that chronic treatment
of even lower doses will elicit an effect over time. However, questions would
arise of the mechanism: 1) Are the drugs acting through their receptors
(DCSNMDAR and PIOPPARγ), 2) Are they acting through receptor
independent pathways (mitochondrial actions), or more likely 3) Are they acting
through both receptor dependent and independent pathways?
This current study observed the receptor independent actions of the drug
combinations and its effects on improving mitochondrial function after the TIC
injury. Another limitation of this study is that only State III, State IV, and the RCR
were conducted for mitochondrial dysfunction in the Seahorse assay.
Alternatively, future studies could determine specifically if complex I is
dysfunctional by measuring the NADH/NAD+ ratio before and after nerve injury.
Since complex I is important for State III and State IV electron transport
respiration, this would be a beneficial experiment to run in order to determine if
128
the mitochondrial dysfunction that is observed is due to alteration of the complex
I redox state (Pandya et al 2013; Sauerbeck et al 2011a). Furthermore, the
activity of the remaining complexes in the mETC could be observed in future
studies using the Oxytherm method to determine if one or more complex is
responsible for the mitochondrial dysfunction that occurs after TIC injury in the
mitochondrial cortex (Mustafa et al 2010; Sullivan et al 2004b).
Since there are no changes observed in the calcium dynamics of the
isolated mitochondria (cortex and brainstem), measuring total oxidative stress
could also provide explanation for the mitochondrial dysfunction observed. By
measuring reactive oxygen/nitrogen species using spectrophotometric assays
along with the H2O2 production (Sullivan et al 2004b), we could not only
determine if oxidative stress is occurring in the mitochondria, but what particular
oxidative stress species is responsible. Taken together with the experiments
listed above, a mechanism for mitochondrial dysfunction could be deduced.
Furthermore, future studies could determine more specifically how the
DCS/PIO drug combination is acting on the mitochondria to reverse dysfunction.
It could be from any of the mechanisms listed above and should be studied in
more detailed. Future studies could analyze the cortical mitochondria from the
mice with TIC treated with chronic feeding of the DCS/PIO combination to
observe if there is greater reversal of mitochondrial dysfunction.
129
5.5. Significance and Innovation
Overall, these studies advanced the field of orofacial pain by providing:
1) Detail characterization of a novel model useful as a tool in identifying
molecular targets that play a major role in the etiology and
maintenance of trigeminal neuropathic pain.
2) First report that identifies upregulated PPARγ in the TBSNC after
injury.
3) First report that PIO attenuates trigeminal neuropathic pain through
activation of PPARγ receptor signaling.
4) First report that the DCS/PIO combination attenuates trigeminal pain
as well as anxiety related to pain.
5) First demonstration that mitochondrial dysfunction occurs in the cortex
of the mice after TIC injury and that this dysfunction can be improved
by the DCS/PIO combination.
The characterization of the TIC injury model is an advancement in the field
of trigeminal pain research because orofacial pain mouse models previously
described do not have characteristics as closely matching those of patients with
chronic facial neuropathic pain (Bornhof et al 2011; Luccarini et al 2006). Since
this model has stronger translational features than the other models display, the
TIC injury model can be used to study pharmaceutical agents that perhaps can
help relieve facial pain and anxiety-related to pain. Finally, since this model is
induced in mice, the model could be used for genetic studies of trigeminal
130
neuropathic pain. Developing specific gene knockout mice will be helpful in
determining other molecular targets that are important in the etiology and
maintenance of trigeminal neuropathic pain.
These studies will be useful when treating orofacial pain patients in the
future. By determining that PPARγ is a molecular target and that PPARγ
activation will attenuate trigeminal pain, the potential exists to repurpose the FDA
approved diabetic therapeutic drug, PIO. It is true that caveats exist when
discussing the possibility of using PIO as a chronic therapeutic option. Currently,
PIO has been withdrawn from the market in Germany and France due to
increased health factors including bladder cancer, edema, and cardiac issues
(Kostapanos et al 2013). However, it is important to mention that clinical and
animal studies performed in non-diabetic subjects that were given chronic
treatments of PIO did not suffer from the same side effects (i.e. no change in LDL
levels and no overt signs of edema and cardiac risk) (Aithal et al 2008; Fullert et
al 2002; Szapary et al 2006). This leads to strong speculation that PIO may not,
in fact, be as toxic in patients who suffer from a metabolic disorder.
Furthermore, although the doses that were used in this study were high,
when comparing the mouse doses to human doses, they were in normal range
prescribed for patients. Clinical dose for PIO ranges from 15 mg/kg to 45 mg/kg /
day. Shaw and colleagues determined a relatable equation factoring the body
weight and surface area of an average mouse and human that better estimates
that the 300 mg/kg PIO dose in mice is equivalent to about 25 mg/kg PIO for a
human(Reagan-Shaw et al 2008). Therefore, this highest dose given to the mice
131
in this study, falls within normal dosage range for humans. Whether this dose will
be efficacious in treatment of the pain that is suffered by patients with chronic
orofacial pain will need to be determined. Furthermore, although DCS is being
tested for the treatment of sciatic pain and the anxiety-related to pain in humans
(Davis et al 2006; Millecamps et al 2007; Norberg et al 2008), this data support
the repurposing of DCS for the treatment of trigeminal neuropathic pain,
specifically in combination with PIO.
Furthermore, new drug therapies/deliveries can be challenged. For
example, a clinical trial could be conducted with the combination of both
DCS/PIO could determine an effective dose combination that would reduce the
side effects of PIO while potentiating the inhibitory effect on neuropathic pain and
the anxiety-related to pain. Many patients will often receive high doses of
medication, or sometimes two pills, an analgesic and an anti-depressant drug
(De Leeuw 2008; Zakrzewska 2013). However, this study determines that
combination of the two, as a double edged sword, act as both an analgesic and
anti-depressant (see chapter 4).
The two drugs together could propel the chronic pain field forward for many
reasons. One being it challenges the way the clinicians, basic scientists, and
industry drug developers think about old drugs. If there is a way to save money
by utilizing available drugs, that not only attenuate pain, but together have a
potentiated effect, then the money invested in drug development could be better
spent and discovery shifted into drug repurposing. This will save time and money
in the long run while treating patients more quickly. Furthermore, the present
132
study supports dual therapy treatment. Recent studies have observed the
synergistic effects of PIO with pravastatin to decrease inflammation (Wei et al
2007) as well as the synergistic effects of DCS and valproic acid for the
extinction of fear (Kuriyama et al 2011).
Finally, this was the first study to determine that mitochondrial dysfunction
is a key play in trigeminal neuropathic pain, highlighting the mitochondria also is
a drug target for treating facial pain. This study also determined ex vivo that the
combination of DCS/PIO is acting independently of their known receptors to
improve mitochondrial dysfunction. Studies have linked mitochondrial dysfunction
to neuronal injury and neuropathy (Bouillot et al 2002; Ferrari & Levine 2010;
Joseph et al 2004; Joseph & Levine 2006; Kim et al 2004; Pandya et al 2007;
Park et al 2006; Schwartz et al 2009; Shin et al 2003; Sui et al 2013; Sullivan et
al 2007), and others to depression (Gardner & Boles 2008; Kato 2011; Kato &
Kato 2000; Rezin et al 2009). This was the first study to determine that a drug
combination targeting mitochondria can in fact attenuate both of these aspects of
pain in vivo. Perhaps these studies will not only encourage a more in depth study
of mitochondria and its link to trigeminal neuropathic pain, but also that the drug
combination itself can be utilized in so many different diseases related to
mitochondrial dysfunction. This suggests other drugs that target the improvement
of mitochondrial dysfunction could be utilized. Future studies need to be
conducted to confirm the specific pathway(s) that DCS/PIO are utilizing to elicit
these effects.
133
5.6. Conclusion
This study was successful in characterizing a novel orofacial neuropathic
pain mouse model, TIC, and determining the usefulness of the similarities it has
with the clinical syndrome of continuous trigeminal neuropathic pain. This study
supports the repurposing of PIO and DCS as useful pharmaceuticals in treating
trigeminal neuropathic pain, particularly in a low dose combination by acting to
improve mitochondrial dysfunction. Overall, this study accentuates the need for
new pharmaceutical treatments for patients suffering with trigeminal pain and
provides two novel molecular targets (PPARγ and mitochondria) as options to
attenuate continuous trigeminal neuropathic pain.
134
Appendix 1. Abbreviations
AAALAC
Association for Assessment and Accreditation of
Laboratory Animal Care International
AAPM
the American Academy of Pain Medicine
ACC
Cingulate cortex
ADP
Adenosine diphosphate
ANOVA
Analysis of variance
ATPase
Adenylpyrophosphatase
BOLD
Blood-oxygen-level dependent
CCI-ION
Chronic constriction injury of the infraortibal nerve
CBT
Cognitive behavioral therapy
2,4-DNP
2,4-Dinitrophenol
DCS
D-cycloserine
DMSO
Dimethyl sulfoxide
EDP
an energy-dissipating pathway
EGTA
Ethylene glycol tetraacetic acid
FDA
Food and Drug Administration
FCCP
Trifluorocarbonylcyanide Phenylhydrazone
fMRI
Functional magnetic resonance imaging
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
IASP
the International Association for the Study of Pain
ION
The infraortibal nerve
IACUC
Institutional Animal Care and Use Committee
135
i.p.
Intraperitoneal
IL-6
Interleukin 6
mETC
the mitochondrial electron transport chain
MIB
Mitochondrial isolation buffer
MRB
Mitochondrial running buffer
NF-κB
Nuclear factor kappa-light-chain-enhancer of
activated B cells
NMDA
N-Methyl-D-aspartate
NIH
National Institute of Health
OCR
Oxygen Consumption Ratio
PAG
Periaqueductal gray
pCCI-ION
Partial chronic constriction injury of the infraortibal
nerve
PFC
Prefrontal cortex
PIO
Pioglitazone
P.O.
per os
PMF
Proton motive force
PPAR-α
Peroxisome proliferator-activated receptor alpha
PPAR-β/δ
Peroxisome proliferator-activated receptor beta/delta
PPARγ
Peroxisome proliferator-activated receptor gamma
PPRE
Peroxisome proliferator-activated receptor response
elements
PPC
posterior parietal cortex
136
ROS
Reactive oxygen species
RCR
the Respiratory Control Ratio
RXR
Retinoid X receptor
S1/S2
Somatosensory cortex 1 and 2
SMA
Insula cortex, supplementary motor area
S.C.
Subcutaneous
TBSNC
Trigeminal brainstem sensory nuclear complex
TIC
Trigeminal Inflammatory Compression
TNF-α
Tumor necrosis factor alpha
TZD
Thiazolidinedione
USDA
the United States Department of Agriculture
VPM
Ventroposteromedial nucleus of the thalamus
137
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151
VITA
DANIELLE LYONS
Birth Place: Louisville, KY
Hometown: Simpsonville, KY
EDUCATION
PhD, candidate
PRESENT
University of Kentucky, College of Medicine, Department of Physiology
Dissertation Title: “Attenuating Trigeminal Neuropathic Pain by
Repurposing Pioglitazone and D-cylcoserine in the Novel Trigeminal
Inflammatory Compression Mouse Model”
Committee: Karin Westlund High (chair), Reny DeLeeuw, Brad Taylor,
Greg Gerhardt, Greg Frolenkov
BS
University of Kentucky, Biology
May 2010
Graduated Cum Laude
Honors Program
Minored in Family Studies
HONORS AND AWARDS
Physiology Teaching Certificate
2014
Honoree participant for the Three Minute Thesis Competition
at the Conference of Southern Graduate Schools
2014
1st place winner of University of Kentucky’s Three Minute Thesis
Competition
2013
Governor’s Scholar Award, Commonwealth of Kentucky
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2006-2010
Valedictorian Scholarship Award
2006
RESEARCH EXPERIENCE
Dissertation Project, University of Kentucky, Lexington, KY
2010-present
Advisor: Karin Westlund High, PhD, Professor of Physiology
Graduate Student Research Rotations,
University of Kentucky, Lexington, KY
2010 to 2011
Brad Taylor, PhD, Professor of Physiology
Greg Gerhardt, PhD, Professor of Anatomy and Neurobiology
Undergraduate Research, University of Kentucky, Lexington, KY 2008 to 2010
Lab Tech, Bernhard Hennig, PhD, RD Professor of Nutritional
Sciences
Lab Undergraduate Research Assistant, Thomas Zentall, PhD,
Professor of Psychology
PUBLICATIONS
Journal Publications
Ma F, Zhang L, Lyons D, Westlund KN. Orofacial Neuropathic Pain Mouse
Model Induced by Trigeminal Inflammatory Compression (TIC) of the Infraorbital
Nerve. Molecular Brain (2012), (5): (4).
Journal Papers Submitted
Lyons, DN. Kniffin, TK. Zhang, L. Ma, Fei, Danaher, R., Miller, C., Carlson, C.,
Westlund, KN. Working Title: Induced Facial Pain, Anxiety-and Depression
Related Behaviors Associated with Trigeminal Inflammatory Compression (TIC)
Injury
Journal Papers In Progress
Lyons, DN., Zhang, L., Ma, Fei, Westlund, KN. Working Title: Rapid Effects of
PPAR-agonists on Alleviation of Orofacial Pain.
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Kniffin, TK, Lyons, DN. Working Title: Review: Orofacial Rodent Models.
Lyons, D, Kniffin, TK, Zhang L, Ma, F, Danaher, R., Miller, C., Carlson, C.,
Westlund KN. Working Title: Low-dose Combination of Pioglitazone and Dcycloserine Attenuated Orofacial Pain by Acting as a Mild Mitochondrial
Uncoupler.
Abstract-Published
Lyons, D, Kniffin, TK, Zhang L, Ma, F, Danaher, R., Miller, C., Carlson, C.,
Westlund KN. Stopping Orofacial Pain: Repurposing Old Drugs for New Use.
Society for Neuroscience. Washington, D.C., 2014
Lyons, D, Kniffin, TK, Zhang L, Ma, F, Danaher, R., Miller, C., Carlson, C.,
Westlund KN. Stopping Orofacial Pain: Repurposing Old Drugs for New Use.
Bluegrass American Physiology Society. University of Louisville, 2014.
Lyons, D, Kniffin, TK, Zhang L, Ma, F, Danaher, R., Miller, C., Carlson, C.,
Westlund KN. Stopping Orofacial Pain: Repurposing Old Drugs for New Use.
Bluegrass Society For Neuroscience. University of Kentucky, 2014.
Brown, A.T., Kline, R.H, Lyons, D. Ighodaro, E.T., Nelson, P, Kilgore, M.,
McGillis, J., Danaher, R., Westlund, KN. Characterization of Cortical pERK
Expression After Trigeminal Inflammatory Compression (TIC) of the Infraorbital
Nerve. Bluegrass Society For Neuroscience. University of Kentucky, 2014.
Lyons, D, Zhang, L., Ma. F., Westlund, KN. Stopping the TIC: Reducing
Trigeminal Neuropathic Pain Using PPAR Agonists. Bluegrass Society For
Neuroscience. University of Kentucky, 2013.
Lyons D, Miller H, Reeves R, Zentall T. Dogs Understanding Human Cues.
Undergraduate Research Day, University of Kentucky, 2009.
PRESENTATIONS AND INVITED LECTURES
Poster Presentation “Stopping Orofacial Pain: Repurposing Old Drugs for New
Use.” Bluegrass American Physiology Society. University of Louisville, 2014.
Poster Presentation “Stopping Orofacial Pain: Repurposing Old Drugs for New
Use.” Bluegrass Society For Neuroscience. University of Kentucky, 2014.
154
Podium Presentation. “Stopping Orofacial Pain.” Three Minute Thesis
Competition. Conference of Southern Graduate Schools. San Antonio, Texas,
2014.
Podium Presentation. “Determine the efficacy and safety of pharmacological
treatments for novel models of orofacial neuropathic pain.” COBRE Review
Board, University of Kentucky, 2014.
Workshop, “Elevator Pitches,” Chalk Talks, Physiology Department, University
of Kentucky, February 2014.
Podium Presentation.. Stopping Orofacial Pain. Three Minute Thesis
Competition. University of Kentucky, 2013.
Podium Presentation. “Stopping the TIC: Alleviating Trigeminal Neuropathic
Pain Using PPAR Agonists.” Physiology Departmental Seminar, University of
Kentucky, 2013.
Guest Speaker “Graduate Research at University of Kentucky.” Asbury
University, Wilmore Ky. April 2013,2014.
PROFESSIONAL AFFILIATIONS
Society for Neuroscience, April 2014-Present
Bluegrass American Physiology Society, March 2014-Present
TEACHING EXPERIENCE
University of Kentucky, Lexington, KY
Teaching Assistant, Department of Physiology, PGY 207
Asbury University, Wilmore, KY
Guest Lecturer, BIO 352: Human Physiology
COMMUNITY SERVICE
Lexington Medical/Dental Refuge Clinic
Southland Christian Church
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