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이학박사학위논문
통증 조절을 위한 다각적 접근:
국소 진통제, 약침 요법 및 시간 제한 섭식에
관한 연구
Multimodal approach for pain management:
Local analgesics, Pharmacopuncture and Time-
restricted feeding
2020 년 2 월
서울대학교 대학원
자연과학대학 뇌인지과학과 전공
이 정 윤
1
ABSTRACT
Multimodal approach for pain management:
Local analgesics, Pharmacopuncture and Time-
restricted feeding
Jeong-yun Lee
Brain Cognitive Neuroscience
The Graduate School
Seoul National University
Pain is defined as an unpleasant sensory and emotional experience
associated with actual or potential tissue damage by International Association for
the Study of Pain (IASP). Since pain has both sensory-discriminative and
affective-motivational dimensions, it is difficult to effectively modulate pain with
a conventional pharmacological approach (NSAIDs and opioids) alone. In an
effort to explore multimodal approaches to pain management, I have focused on
the therapeutic effect of local analgesics (sinomenine), pharmacopuncture
(acupoint stimulation with drug, Scolopendra subspinipes) and time-restricted
feeding (fasting/refeeding) on animal pain models as below.
In the first chapter, I revealed that sinomenine can produce peripheral
analgesic effect by reducing cellular excitability of nociceptive neurons. The
intraperitoneal injection of sinomenine suppressed formalin-induced pain
behavior and c-Fos expression. Sinomenine inhibited voltage-gated sodium
2
current in nociceptive neurons. Furthermore, the intraplantar injection of
sinomenine also suppressed pain behavior. Therefore, sinomenine could be a
potential pharmacological therapeutic agent for local anesthesia and analgesia.
In the second chapter, I demonstrated that acupoint stimulation with
Scolopendra subspinipes (pharmacopuncture with Scolopendra subspinipes, SSP)
produces an analgesic effect via alpha2-adrenoreceptors in the spinal cord. SSP
into Zusanli acupoint had a significant analgesic effect on oxaliplatin-induced
neuropathic pain model. The intrathecal injection of yohimbine (alpha2-
adrenoeceptor antagonist) reversed SSP-induced analgesic effect. Therefore, SSP
produces an analgesic effect by activating descending pain inhibitory system,
which suggests a possibility for pain management.
In the third chapter, I focused on the analgesic effect of fasting and
refeeding. Both 24h fasting and 2h refeeding produce analgesic effect but via
different mechanisms, suggesting that endocannabinoid and opioid system is
only involved in fasting-induced analgesia, whereas refeeding-induced analgesia
is associated with eating behavior and calorie recovery effect. Therefore, the
regulation of feeding (time-restricted feeding) may have abundant benefits to
modulate pain and might provide a novel strategy for the management of pain.
Key Words: pain; sinomenine; voltage-gated sodium channel; Scolopendra
subspinipes; pharmacopuncture; alpha2-adrenoceptors; fasting; time-restricted
feeding
Student Number: 2015-22669
3
CONTENTS
Contents ............................................................................................................... 3
List of figures ....................................................................................................... 5
Background .......................................................................................................... 7
Purpose ............................................................................................................... 22
CHAPTER 1:The analgesic effect of sinomenine via inhibition of voltage-
gated sodium channel ...................................................................................... 23
Abstract ...................................................................................................... 24
Introduction ................................................................................................ 25
Materials and Methods ............................................................................... 26
Results ........................................................................................................ 31
Discussion .................................................................................................. 44
CHAPTER 2: Pharmacopuncture with scolopendra subspinipes suppresses
mechanical allodynia in oxaliplatin-induced neuropathy: Mediation by
spinal alpha2-adrenoceptor ............................................................................. 48
Abstract ...................................................................................................... 49
Introduction ................................................................................................ 50
Materials and Methods ............................................................................... 51
Results ........................................................................................................ 55
Discussion: ................................................................................................. 65
CHAPTER 3:Both acute fasting and refeeding alleviate inflammatory pain
but via different pathways ............................................................................... 68
Abstract ...................................................................................................... 69
Introduction ................................................................................................ 70
4
Materials and Methods ............................................................................... 72
Results ........................................................................................................ 75
Discussion .................................................................................................. 88
Conclusion ......................................................................................................... 91
References .......................................................................................................... 92
국문초록 ............................................................................................................ 98
5
LIST OF FIGURES
Background
Figure 1. Pain pathway ...................................................................................... 12
Figure 2. Approaches to pain management ....................................................... 14
Figure 3. Local analgesics ................................................................................. 16
Figure 4. The mechanism of ascending nociceptive control ............................. 18
Figure 5. Benefits of time-restricted feeding .................................................... 20
Chapter 1
Figure 6. Effects of sinomenine on formalin-induced pain ............................... 34
Figure 7. Effects of sinomenine on action potentials in small sized dorsal root
ganglion neurons .................................................................................. 36
Figure 8. Effects of sinomenine on voltage-gated sodium channel in small sized
dorsal root ganglion neurons ................................................................ 38
Figure 9. Intraplantar administration of sinomenine on formalin-induced pain
behavior ................................................................................................ 40
Figure 10. Effects of sinomenine in rota-rod test .............................................. 42
Chapter 2
Figure 11. Effect of Scolopendra subspinipes pharmacopuncture (SSP) on
formalin-induced acute inflammatory pain model ............................... 57
Figure 12. Effect of Scolopendra subspinipes pharmacopuncture (SSP) on
oxaliplatin (OXA)-induced neuropathic pain model ............................ 59
6
Figure 13. Peripheral nerve activation of the specific acupoint is associated
with Scolopendra subspinipes pharmacopuncture (SSP)-induced anti-
allodynia ............................................................................................... 61
Figure 14. Effects of pretreatment of intrathecal injection of yohimbine,
naloxone, or methysergide on Scolopendra subspinipes
pharmacopuncture (SSP)-induced anti-allodynia................................. 63
Chapter 3
Figure 15. The effect of fasting and refeeding on acute pain behavior ............. 78
Figure 16. Changes in body weight, blood glucose level and food intake by
refeeding in naïve mice ........................................................................ 80
Figure 17. The analgesic effect of refeeding with non-calorie agar on
inflammatory pain behavior ................................................................. 82
Figure 18. The analgesic effect of intraperitoneal administration of D-glucose
on inflammatory pain behavior ............................................................ 84
Figure 19. The effect of opioid receptor and cannabinoid receptor (CB1)
antagonist on fasting and refeeding-induced analgesia ...................... 86
7
BACKGROUND
1. Pain pathway
Pain is somatic and emotional sensations1. The sensory-discriminative aspect
of pain (nociception) provides sensory information such as location, quality, and
intensity. Emotional pain consists of hedonic aspect (pleasantness/unpleasantness)
and affective-motivation aspect which creates behavior to escape from pain as a
secondary pain effect. Pain pathways are largely divided into three categories:
two ascending pain pathways (the lateral pain pathway for nociception and the
medial pain pathway for emotional pain) and one descending pain pathway (Fig.
1 A)2. In the lateral pain pathway, noxious stimuli activate nociceptive primary
afferent neurons (1st order neuron) which transmit signals from the periphery to
the dorsal horn of the spinal cord (2nd order neurons). The thalamocortical
neurons (3rd order) project from the ventral posterior nucleus of the thalamus to
the primary somatosensory cortex, thus contributing to nociception (Fig.1 B and
C). On the other hand, the medial pain pathway projects from the medial thalamic
nuclei to brain regions including the prefrontal, the anterior cingulate cortex
(ACC) and the insular cortex which are critical for regulation of emotions and
susceptible to various cognitive factors (Fig.1 C)3. While the ascending pain
pathway conveys pain signal from periphery to central cortex, the descending
pain pathway including the ACC, the periaqueductal gray (PAG), and the
rostroventral medial medulla (RVM) modulates pain signal from central cortex
to the spinal cord (Fig.1 D)4. The main neurotransmitters involved in descending
inhibitory pain pathway, are known as serotonin, noradrenalin, and opioid.
8
2. Approaches to pain management
The World Health Organization (WHO) proposed the analgesic ladder
according to pain intensity (Fig. 2A)5,6. The guidelines from the WHO ladder
suggest non-opioid drugs, especially nonsteroidal anti-inflammatory drugs
(NSAIDs) for the first steps in pain therapy and also recommend the adjuvant
treatments (anticonvulsants, antidepressants, alpha2-adrenoceptor agonists, N-
Methyl-D-aspartate receptor antagonists, etc.) for neuropathic pain. In the case
of pain that is not controlled with non-opioid drugs, the use of opioid-based
analgesics is recommended. Although this therapeutic guideline is widely
available for the administration of analgesics, conventional analgesics limited
in pain treatment and many patients still experience pain7. Furthermore, pain is
a multifaceted phenomenon with various etiologies and symptoms. Recently,
integrated pain management is being recognized as an effective care for pain
management (Fig. 2B)8. For example, multimodal analgesia combines various
groups of medications such as local anesthetics, NSAIDs, gabapentin, tricyclic
antidepressants, opioids etc. to produce a synergistic effect. Physical (sensory)
interventions including massage, positioning, hot/cold, acupuncture, and
transcutaneous electrical nerve stimulation (TENS) are also well known to
relive pain9. Furthermore, behavioral intervention and improvement of lifestyle
manage pain better through psychotherapy, sleep, nutrition, mood, and exercise
(active self-care therapies).
9
3. Local analgesics
The most commonly used systemic analgesics have various side effects (Fig.
3A)10, while local analgesics have the advantage of minimizing the side effects
by treating drugs only in actual or potential tissue damage sites11. The mechanism
of local analgesics is largely within the peripheral nervous system. Since the
activity of nociceptive neurons (Aδ and C-fiber) is regulated by ion channels, ion
channels expressed on nociceptive neurons are the key molecular target for
diverse analgesics (Fig. 3B)12. For example, eugenol, the main component in
clove plant, is well known for having local anesthetic actions in periphery by
regulating various ion channels (TRPV1, P2X, voltage-gated sodium channels,
voltage-gated potassium channels, voltage-gated calcium channels, etc.), thereby
providing a pharmacological mode of action for their wide use in dental clinic to
alleviate dental pain13. Especially, voltage-gated sodium channels (VGSCs) are
the main ion channels for generating action potentials, thereby responsible for
transmitting pain signals in the nociceptive neurons14-16. Local analgesics
targeting VGSCs, such as procaine, lidocaine, and mepivacaine, are the most
widely used17. It is also possible to selectively block pain signals by blocking
VGSCs with the permanently charged lidocaine derivative QX-314 that enters
through large-pore ion channels selectively expressed in nociceptive neurons18-20
10
4. Pharmacopuncture
Peripheral stimulation by noxious stimuli is well known to produce an
analgesic effect by activating descending pain pathway (Fig. 4)21,22. Acupoint is
anatomically defined areas on the body surface with unique features such as high
electrical conductance, hypersensitivity, needling sensation, therapeutic effect
and connection with internal organ23,24. Interestingly, recent studies have shown
that these features of acupoint are closely related to Aδ and C-fiber. The high
electrical conductance or hypersensitivity at acupoint is associated with
neurogenic substance P (SP) and calcitonin gene-related peptide (CGRP) from
Aδ and C-fiber24. Primary afferent neurons, especially Aδ and C-fiber, are
essential for mediating the therapeutic effect of acupoint stimulation25,26. It is well
known that the low-frequency electrical stimulation into acupoint produced
analgesic effect by modulating mu-opioid receptor, serotonin receptor, and
alpha2-adrenoceptor in spinal cord. Drug-induced acupoint stimulation, termed
pharmacopuncture, is a method of safely stimulating acupoint for a long time.
Bee venom is the most commonly used drug in pharmacopuncture. In clinical
study as well as various animal pain models, pharmacopuncture with bee venom
(BVP) is known to cause pain but has been shown to have a long-lasting analgesic
effect via serotoninergic and adrenergic system, but not opioidergic system27,28.
11
5. Time-restricted feeding
Hunger is well known as a powerful driving force to change cognition.
Several clinical evidences have shown that therapeutic fasting is effective for
pain relief and mood enhancement regardless of body weight change in chronic
pain patients. In various animal pain models, fasting or calorie restriction also
has an analgesic effect. However, it is recently shown that Time-Restricted
Feeding (TRF) may be more successful than Calorie-Restricted Feeding (CRF)
for not only on healthy lifespan but also on neural circuits related to cognition
and emotion29. Many studies have shown that TRF with high fat diet did not cause
metabolic disease and obesity30 (Fig. 5A). As compared with TRF, Ad libitum
feeding interfere with circadian rhythm resulting in metabolic dysfunction and
metabolic disease. Furthermore, TRF is accompanied by intermittent metabolic
switching such as negative energy balance (fasting and/or exercise) followed by
positive energy balance (eating, resting, and sleeping) (Fig. 5B), which is known
to alter cell signaling in neurons and affect neuroplasticity31. The sensation of
hunger and satiety is closely related to the mesolimbic reward system involving
the regulation of food intake32. Since the unpleasantness of pain as well as the
relief of pain is encoded by mesolimbic circuitry, TRF may have many benefits
to modulate pain perception.
12
Figure 1.
13
Figure 1. Pain pathway
(A) Two ascending pain pathway and one descending pain inhibitory pathway.
(B) Ascending pain pathway consists of three neuron pathways (1st, 2nd, and 3rd
order neurons) (C) Ascending pain pathway is divided into lateral and medial
pathway. Lateral pathway is responsible for detecting painful sensation and for
identifying the location of painful stimulus. Medial pathway is responsible for
the emotional aspect of pain. (D) Descending pain inhibitory pathway suppresses
pain signal in the spinal cord by modulating monoaminergic inputs.
Fig. 1A modified and extended from De Ridder et al. 2016 (2)
Fig. 1B modified and extended from Pearson Education, Inc. 2011
Fig. 1C modified and extended from Bushnell et al 2013 (3)
Fig. 1D modified and extended from Benarroch et al 2008 (4)
14
Figure 2.
15
Figure 2. Approaches to pain management
(A) The WHO Pain Treatment 3 Step Ladder. The stages are divided according
to pain intensity and the use of opioids. (B) Integrated care for pain; non-opioid
and non-pharmacologic treatment. The use of non-opioid medication as medical
interventions is useful for multimodal analgesia which combines various groups
of medications (local anesthetics, NSAIDs, opioids, NMDA agonist, alpha-2
agonists, etc.). Physical (sensory) interventions include massage, positioning,
hot/cold, acupuncture and Transcutaneous Electrical Nerve Stimulation (TENS).
Behavioral intervention and improvement of lifestyle manage pain relief through
psychotherapy, sleep, nutrition, mood, and exercise (active self-care therapies).
Fig. 2A modified and extended from bpacnz pain management (5)
Fig. 2B modified and extended from Conschafter, A 2015(8)
16
Figure 3.
17
Figure 3. Local analgesics
(A) Multimodal analgesic sites of action in the central and peripheral nervous
systems and adverse effects of analgesics commonly used. (B) Ion channels
targeting for local analgesics in nociceptive neurons. Ion channels detecting
noxious thermal, chemical, and mechanical stimuli are expressed in nociceptive
nerve ending. Voltage-gated sodium channels (Nav1.7, Nav1.8 and Nav1.9)
mainly are expressed in nociceptive neuron and play an important role in
generating action potential.
Fig. 3A modified and extended from Pitchon et al. 2018 (10)
Fig. 3B modified and extended from Juárez-Contreras R et al. 2018 (12)
18
Figure 4.
19
Figure 4. The mechanism of ascending nociceptive
control
Peripheral stimulations by noxious stimuli such as electrical, chemical or
acupuncture alter the peripheral nerve activity, which induces the activation of
descending pain inhibitory system. In persistent pain condition, adequate
peripheral stimulation effectively suppresses pain signaling.
Fig. 4 modified and extended from BMJ 2014;348:f7656
20
Figure 5.
21
Figure 5. Benefits of time-restricted feeding
(A) Time-Restricted Feeding (TRF) improves circadian clock and metabolism
rhythms. TRF increase resistance to disease caused by high fat diets. (B)
Metabolic switching enhances neuroplasticity and optimize brain function.
Fig. 5A modified and extended from Hatori, M. et al. 2012 (30)
Fig. 5B modified and extended from Mattson, M. P. et al. 2018 (31)
22
PURPOSE
The standard of care (NSAIDs and opioids) for pain currently has many
limitations. In an ongoing attempt to control pain through a multimodal approach
based on pain mechanisms, I have focused on the therapeutic effect of local
analgesics (sinomenine), pharmacopuncture (acupoint stimulation with drug,
Scolopendra subspinipes), and time-restricted feeding (fasting/refeeding) as
below.
To confirm whether sinomenine produces an analgesic effect on
animal pain behavior and if so, to investigate the mechanism of
sinomenine as a local analgesic.
To investigate the analgesic mechanism of pharmacopuncture with
Scolopendra subspinipes.
To examine whether animal pain behavior is affected by fasting and
refeeding, with a focus on calorie state, and if so, to investigate their
potential mechanism.
23
CHAPTER 1:
The analgesic effect of sinomenine
via inhibition of voltage-gated
sodium channel
*This chapter has been largely reproduced from articles published by
Jeong-Yun Lee, Seo-Yeon Yoon, Jonghwa Won, Han-Byul Kim, Youngnam Kang, Seog
Bae Oh. Sinomenine produces peripheral analgesic effects via inhibition of voltage-
gated sodium currents. Neuroscience, 2017, Sep;358:28-36. doi:10.1016/
24
ABSTRACT
Sinomenium acutum has been used in traditional medicine to treat a
painful disease such as rheumatic arthritis and neuralgia. Sinomenine, which is a
main bioactive ingredient in Sinomenium acutum, has been reported to have an
analgesic effect in diverse pain animal models. However little is known about the
detailed mechanisms underlying peripheral analgesic effect of sinomenine. In the
present study, I aimed to elucidate its cellular mechanism by using formalin-
induced acute inflammatory pain model in mice. I found that intraperitoneal (i.p.)
administration of sinomenine (50 mg/kg) suppressed formalin-induced paw
licking behavior in both the first and the second phase. Formalin-induced c-Fos
protein expression was also suppressed by sinomenine (50 mg/kg, i.p.) in the
superficial dorsal horn of spinal cord. Whole cell patch clamp recordings from
small-sized dorsal root ganglion (DRG) neurons revealed that sinomenine
reversibly increased the spike threshold and the threshold current intensity for
evoking a single spike and decreased firing frequency of action potentials evoked
in response to a long current pulse. Voltage-gated sodium currents (INa) were also
significantly reduced by sinomenine in a dose-dependent manner (IC50 = 2.3 ±
0.2 mM). Finally, I confirmed that intraplantar application of sinomenine
suppressed formalin-induced pain behavior only in the first phase, but not the
second phase. Taken together, these results suggest that sinomenine has a
peripheral analgesic effect by inhibiting INa.
25
INTRODUCTION
Sinomenium acutum has been traditionally used as a herbal medicine to
treat rheumatic arthritis and neuralgia33. Sinomenine is its main bioactive
ingredient in Sinomenium acutum and is well known to have immunosuppressive
and anti-inflammatory effects34. Interestingly, recent studies have also
demonstrated analgesic effects of sinomenine in rodents with nociceptive,
inflammatory and neuropathic pain35. Sinomenine-induced analgesia is reversed
by a GABAA receptor antagonist in postoperative pain and neuropathic pain
models36. Apart from the pathological animal pain models, tail flick test shows
that sinomenine has analgesic effects by activating opioid μ-receptor37. While
these studies have found potential cellular targets for sinomenine in the central
nervous system (CNS)38, little is known about the cellular mechanism or
sinomenine-induced peripheral analgesia.
VGSCs are the main ion channels for transmitting pain signals in the
nociceptive neurons14-16. Thus, VGSCs in primary afferent neurons is the key
molecular target for diverse analgesics including local anesthetics such as
lidocaine17. In chapter 1, I sought to explore the analgesic effect of sinomenine
by using formalin-induced acute pain model and further focused its peripheral
mechanisms by examining its effects on the excitability of nociceptive neurons.
I found that sinomenine can produce peripheral analgesic effect by reducing
cellular excitability of nociceptive neurons and the inhibition of VGSC is likely
to contribute to its action.
26
MATERIALS AND METHODS
Animals
Male C57BL/6 mice weighing 20-28 g were used for the experiment.
They were housed 4-6 per cage at a temperature-controlled room (23 ± 1 , 12
h/12 h light/dark cycle) and maintained with pellet diet and tap water ad libitum.
All surgical and experimental procedures were reviewed and approved by the
Institutional Animal Care and Use Committee (IACUC) at Seoul National
University.
Formalin test
Mice were acclimated in cage at least for a week and then adapted in an
acrylic observation chamber (size ranges 12×12×12 cm) before the experiment
at least three times. A mirror was located at 45° angle below the chamber to
observe the paws. Formalin test was performed by intraplantar (i.pl.) injection of
1% formalin. 20 μl of 1% formalin was injected subcutaneously into the plantar
surface of the right hind paw with a 31-gauge needle of 0.3 ml insulin syringe.
Following formalin injection, the animals were immediately placed in a test
chamber and recorded using a video camera for a period of 40 minutes. The time
mice spent licking or biting was measured during each 5 minutes by an observer
who was blinded to the treatment. Formalin-induced pain behaviors during 0-10
minutes after formalin injection represented the first phase and during 10-40
minutes after formalin injection represented the second phase.
c-Fos immunohistochemistry
Animals were sacrificed 2 h after formalin injection for analysis of c-Fos
27
proteins. Animals were anaesthetized by intraperitoneal (i.p.) injection of
pentobarbital (60 mg/kg) and transcardially perfused with 0.01 M phosphate-
buffered saline (PBS) including 500 U/L heparin followed by 4%
paraformaldehyde (PFA) in 0.01 M PBS. The spinal cord was post-fixed by 4%
PFA overnight and transferred to 30% sucrose in 0.01 M PBS for 2 - 3 weeks.
Frozen specimen was transversely sectioned into 30 μm with cryotome and
sections were stored in cryoprotectant at -20 . Free floating sections were
washed with 0.01 M PBS and incubated in 0.3% H2O2 (in distilled water) for 30
minute at room temperature (RT). After elimination of endogenous peroxidase,
sections were washed with 0.01 M PBS and Pre blocked with 5% normal goat
serum (NGS) in PBS with 0.3% triton x (PBST) for 1 h at RT. Sections were
incubated in 1st antibody (PC38, Calbiochem, USA; 1:1000 in 1% NGS (in 0.3%
PBST)) for 48 h at 4 , washed with 0.01 M PBS to remove 1st antibody and
incubated in biotinylated goat anti rabbit (BA1000, Vector laboratories, USA;
1:400 in 0.01 M PBS) for 2 h at RT. Sections were processed with ABC kit
(PK-6100, Vectastain ABC kit, Vector laboratories, USA), visualized with DAB
kit (DAB substrate kit for peroxidase, Vector laboratories, USA) and then
mounted on slide glass. After air drying, all sections were cover slipped with
mountant (H-1000, Vectashield mounting medium, Vector laboratories, USA).
Cell counting and image analysis
For the quantification of c-Fos expression, I confirmed that most c-Fos
expression induced by injection of 1% formalin (i.pl.) localized in Lumbar (L) 4-
5 segment. Four sections with highest expression of c-Fos per animal were
chosen and superficial dorsal horn (lamina I-II) of L4-5 segments were selected
for analysis. The number of c-Fos positive neurons was counted blindly and the
28
mean value was used as representative counts. Using image J, the image of
selected sections was converted to grey scale, background subtracted, enhanced
and sharpened. Intensity threshold was adjusted and then analyzed.
Dorsal root ganglion (DRG) culture
Mice were sacrificed by exposure to isoflurane and decapitated. DRG
were isolated in cold HBSS (Welgene, Korea) and incubated in 3 ml HBSS
containing 1 mg/ml collagenase (Roche, USA) / 2.4 U/ml dispase II (Roche, USA)
for 1 h at 37 . Then DRG were digested by 0.25% trypsin in PBS for 7 min at
37 , inactivated by 2 ml 0.25% trypsin inhibitor (Sigma, USA) and 2 ml
DMEM containing 10% fetal bovine serum (Gibco, USA) / 1% pen strep (Gibco,
USA) and washed by 4 ml DMEM (FBS / pen strep). DRG neurons were
triturated using fire-polished pasteur pipette to separate cells. The cells were
resuspended in neurobasal medium (Gibco, USA) containing B-27 supplement
(Gibco, USA), 1% pen strep, L-glutamine (1 mM), placed on cover slips coated
with poly-D-lysine (Sigma, USA). After 1h cells were fed with fresh neurobasal
medium (B-27 supplement / pen strep / L-glutamine) and maintained in a 5%
CO2 - 95% O2 incubator at 37 °C.
Electrophysiological recordings
Whole-cell current- and voltage-clamp recordings were performed in
small-sized DRG neurons (<20 μm diameter) at RT with an Axopatch 200B
amplifier to record action potentials (APs) and INa. Data were sampled at 10 kHz.
The patch pipettes were pulled from borosilicate capillaries. When filled with
following pipette solutions, their resistances were 2-6 MΩ. A -10 mV liquid
junction potential was corrected. The extracellular solution was driven by gravity
29
and continuously perfused (1-2 ml/min). The pipette solution for current clamp
contained (in mM): K-gluconate 140, CaCl2 1, MgCl2 2, EGTA 10, K2ATP 5,
HEPES 10, adjusted to pH 7.4 with KOH, osmolality 300 mOsm. Extracellular
solution for current clamp contained (in mM): NaCl 140, HEPES 10, CaCl2 2,
MgCl2 1, glucose 10, KCl 5, adjusted to pH 7.4 with NaOH, osmolality 300
mOsm. Single APs were evoked by 5 ms depolarizing current pulses. The spike
threshold was measured at the onset of the positive deflection of the first
differentiation of the spike potential. A series of APs were evoked by 500 ms
depolarizing current pulses. The pipette solution for voltage clamp contained (in
mM): CsCl 100, sodium L glutamic acid 5, TEACl 30, CaCl2 0.1, MgCl2 2, EGTA
11, HEPES 10 adjusted to pH 7.4 with CsOH osmolality 300 mOsm.
Extracellular solution for voltage clamp contained (in mM): NaCl 140, HEPES
10, CaCl2 2, MgCl2 1, glucose 10, KCl 5, adjusted to pH 7.4 with NaOH,
osmolality 300 mOsm. The INa was evoked by a test pulse stepped to -10 mV
from a holding potential of -90 mV every 20 s.
Rota-rod test
Mice were placed on the horizontal bar rotating at a speed of 4 r.p.m
using rota-rod apparatus (DJ-4009, Dae-Jong Engineering &Clean Technology,
Seoul, Korea). Twenty-four hours before the actual rota-rod test all mice were
tested and those that were able to remain on the rod for at least 120 s were
included in the study. Performance time on the bar (in sec) and number of falls
over a 2 minutes were measured. The scores were then compared and analyzed 5
minutes (PRE) before and 30 minutes (POST) after treatment with vehicle or
sinomenine. The test was repeated 3 times and the mean value for each animal
was recorded.
30
Administration of sinomenine
Sinomenine hydrochloride was purchased from TCI chemicals (Tokyo,
Japan). In the formalin test, to examine the systemic effects of sinomenine,
sinomenine was diluted in normal saline and intraperitoneally injected at a dose
of 25 mg/kg, 50 mg/kg or 75 mg/kg in a volume of 10 ml/kg body weight 30
minutes prior to the formalin injection. Control animals received (i.p.) an
equivalent volume of vehicle. To evaluate the peripheral effects of sinomenine,
formalin test was performed after i.pl. injection of sinomenine (500 μg per mouse)
which was diluted in 1% formalin (20 μl/saline). For electrophysiological
recordings, sinomenine was dissolved in the extracellular solutions to their
concentration. The solution was driven to cell by gravity and continuously
perfused (1-2 ml/min).
Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 6.0
(GraphPad Software, USA). Comparison between two groups was made using
the unpaired or paired Student's t-test to analyze the expression of c-Fos,
electrophysiological recordings of APs, formalin test and rota-rod test. For
multiple comparisons, data were analyzed using the one-way ANOVA followed
by Tukey’s test to determine formalin test and electrophysiological recordings of
INa. Data are presented as mean ± SEM. Differences with p<0.05 were considered
significant.
31
RESULTS
The effect of sinomenine on formalin-induced pain behavior
I investigated the analgesic effect of sinomenine using formalin model as
it is a reliable model for testing the analgesic effect and mechanism of a drug39-
41. Injection of 1% formalin (i.pl.) elicited typical biphasic pain behavior (Fig.
6Aa). As compared with the vehicle group, 25 mg/kg (i.p.) sinomenine did not
induce statistically significant decrease in formalin-induced pain behavior (Fig.
6Ab). However, sinomenine treated group (i.p.) had a significant analgesic effect
in both the first phase and second phase, at the dose of 50 mg/kg and 75 mg/kg
(Fig. 6Ab). In the first phase, formalin-induced pain behavior was reduced by 45
± 9.8% at the dose of 50 mg/kg (*p<0.05) and 81.4 ± 5.1% at the dose of 75
mg/kg, respectively (***p<0.001). In the second phase, formalin-induced pain
behavior was reduced by 38.5 ± 6.8% at the dose of 50 mg/kg (**p<0.01) and
96.3 ± 2% at the dose of 75 mg/kg, respectively (***p<0.001).
The expression of c-Fos protein is a measure of acute nociception
following noxious stimulation42. Thus, it is commonly used to show the analgesic
property of the drug43. Injection of 1% formalin (i.pl.) caused high c-Fos
expression in superficial dorsal horn innervated by L4-5 DRG neurons (Fig. 6B).
As compared with the vehicle group, sinomenine (50 mg/kg i.p.) reduced the
number of c-Fos positive neurons in superficial dorsal horn by 32.1 ± 10.1%
(*p=0.0292) (Fig. 6B).
Collectively, these results showed that sinomenine produces an analgesic
effect in acute inflammatory pain, suggesting that peripheral nociceptive neurons
might be targeted by sinomenine.
The effect of sinomenine on APs and VGSCs in small-sized DRG neurons
32
I next investigated the effects of sinomenine on the excitability of small-
sized DRG neurons (<20 μm diameter). 1mM sinomenine increased the spike
threshold from –31 ± 1.6 mV to –26.6 ± 2.1 mV (**p=0.0023) and the current
threshold by 21.5 ± 5% (**p=0.0032) (Fig 6A), and also significantly decreased
AP amplitude by 18.1 ± 3.2% (**p=0.0013) and AP overshoot level by 28.1 ±
4.9% (**p=0.0021) (Fig. 7A). Concomitantly, half- width of AP was increased
by 89.3 ± 29.9% (**p=0.0057) (Fig. 7A). When I examined the effect of
sinomenine on a series of APs, I found that AP firing frequency was also
decreased by 3 mM sinomenine and restored partially after washout (Fig. 7B).
Compared with the control, 3 mM sinomenine reduced AP firing frequency by
39.5 ± 6% (**p=0.007).
Because the VGSCs are responsible for the generation of the APs, I tested
the effect of sinomenine on INa in small-sized DRG neurons by a command pulse
stepped from -90 to -10 mV. As compared with the control, 3 mM sinomenine
significantly inhibited INa in small-sized DRG neurons by 63.3 ± 3.2%
(***p<0.001) (Fig. 8A). INa was decreased by sinomenine in a dose-dependent
manner with 0.1 mM by 7.7 ± 1%, 0.3 mM by 13 ± 2%, 1 mM by 29.9 ± 2.3%,
3 mM by 63.3 ± 3.2% and 10 mM by 83.3 ± 3.2%. (Fig. 8B, IC50 = 2.3 ± 0.2
mM).
These results suggest peripheral action of sinomenine on VGSCs in
nociceptive neurons, which might be associated with analgesic effect of
sinomenine on acute inflammatory pain.
Intraplantar application of sinomenine on formalin-induced pain behavior
To clarify the peripheral action of sinomenine, I then examined whether
peripheral injection of sinomenine indeed produces analgesic effects. I found that
33
Injection of 500 μg sinomenine (i.pl.), co-administrated with 1% formalin, only
reduced the first phase of formalin-induced pain behavior by 38.2 ± 5.9%
(**p=0.0057), whereas the second phase of formalin-induced pain behavior was
not affected by sinomenine (Fig. 9).
The effect of sinomenine on motor function in rota-rod test
Because drug-induced motor function impairment may result in false
positive results in nociceptive pain behavior, I carried out rota-rod test to examine
the effect of sinomenine on motor function. I did not observe motor activity
impairment at the dose of 25 mg/kg and 50 mg/kg, whereas there was a
significant motor dysfunction at the dose of 75 mg/kg (Fig. 10). 75 mg/kg
sinomenine (i.p.) significantly increased number of falls (*p=0.0267) and
decreased performance time (***p=0.0002) in rota-rod test.
34
Figure 6.
35
Figure 6. Effects of sinomenine on formalin-induced
pain
(A) Effects of sinomenine (SIN) on the time course curves of formalin-induced
pain behavior (a). Formalin-induced pain behavior was divided into two phases
and analyzed (b). The bar graph represented first phase (0-10 min) and second
phase (10-40 min) of formalin-induced pain behavior. Intraperitoneal (i.p.)
injection of sinomenine inhibited formalin-induced pain behavior in both first
phase and second phase at the dose of 50 mg/kg or 75 mg/kg. *p<0.05, **p<0.01
and ***p<0.001 (one-way ANOVA followed by Tukey’s test) (B) Intraplantar
injection of 1% formalin induced c-Fos expression in superficial dorsal horn of
Lumbar 4-5 (L4-5) segments (a). Sinomenine (SIN, 50 mg/kg, i.p.) decreased
formalin-induced c-Fos expression in superficial dorsal horn of L4-5 (b). The
quantification of c-Fos expression (c). 50 mg/kg sinomenine suppressed
formalin-induced c-Fos expression in superficial dorsal horn of L4-5 as
compared with vehicle. *p<0.05 (unpaired t test, two-tailed), Scale bar = 200 μm,
SDH; superficial dorsal horn
36
Figure 7.
37
Figure 7. Effects of sinomenine on action potentials in
small sized dorsal root ganglion neurons
(A) The inhibitory effect of sinomenine on action potentials (APs). A
representative APs trace (a). 1 mM sinomenine (SIN) increased the spike
threshold (b) and the current threshold (c), and inhibited AP amplitude (d) and
peak (e). After application of 1 mM sinomenine, half-width of AP was increased
(f). (B) sinomenine decreased APs firing frequency. Series of APs were recorded
during 500 ms depolarizing current step. 3 mM sinomenine inhibited APs firing
frequency (n=4). Series of APs returned partially after washout. **p<0.01 (paired
t test, two-tailed)
38
Figure 8.
39
Figure 8. Effects of sinomenine on voltage-gated sodium
channel in small sized dorsal root ganglion neurons
(A) 3 mM sinomenine (SIN) inhibited voltage-gated sodium current (INa). Time
course of effects of 3 mM sinomenine on INa (1) before, 2) during, 3) after the
application of 3 mM sinomenine) (a). INa trace at the points indicated (b).
Normalized INa. As compared with control 3 mM sinomenine significantly
decreased INa (n=6) (c). ***p<0.001 (repeated measures ANOVA followed by
Tukey’s test) (B) INa was decreased by sinomenine in a dose dependent manner
with IC50 = 2.3 ± 0.2 mM. (0.1 mM n=4, 0.3 mM n=5, 1 mM n=8, 3 mM n=6,
10 mM n=5)
40
Figure 9.
41
Figure 9. Intraplantar administration of sinomenine on
formalin-induced pain behavior
(A) Effects of sinomenine (SIN) on the time course curves of formalin-induced
pain behavior. (B) Formalin-induced pain behavior was divided into two phases
and analyzed. The bar graph represented first phase (0–10 min) and second phase
(10–40 min) of formalin-induced pain behavior. Intraplantar injection of
sinomenine (500 μg) inhibited formalin-induced pain behavior only in the first
phase. **p<0.01 (unpaired t test, two-tailed)
42
Figure 10.
43
Figure 10. Effects of sinomenine in rota-rod test
The performance time on rota-rod (A) and the number of falls (B) was measured
5 minutes (PRE) before and 30 minutes (POST) after intraperitoneal (i.p.)
injection with vehicle or sinomenine. Sinomenine had no effect on rota-rod test
at the dose of 25 mg/kg (n=6) or 50 mg/kg (n=7). 75 mg/kg sinomenine showed
significant difference in rota-rod score (n=6). *p<0.05 and ***p<0.0001 (paired
t test, two-tailed)
44
DISCUSSION
In chapter 1, I show peripherally mediated-analgesic action of sinomenine.
I found that not only i.p application, but also i.pl. application, of sinomenine
produces analgesic effect on formalin-induced pain model. Sinomenine inhibited
formalin-induced c-Fos expression in the superficial dorsal horn of spinal cord.
Using whole-cell patch clamp recordings, I also found that sinomenine reduces
cellular excitability by inhibiting INa in small-sized DRG neurons.
The formalin-induced pain model in rodents typically show biphasic pain
behavior, each caused by different mechanisms40,41,44. The first phase occurs due
to direct stimulation of nociceptor in C-fiber and the second phase is a pain
response caused by peripheral inflammation and functional changes in central
processing44. In the present study, i.p. injection of sinomenine significantly
decreased formalin-induced pain behavior in both first phase and second phase.
However, i.pl. injection of sinomenine decreased formalin-induced pain behavior
only in the first phase (Fig. 6 and Fig. 9). These results suggest that sinomenine
has peripheral analgesic mechanism.
The analgesic effects of sinomenine via i.p. route on the second phase
might result from its central mechanisms. Recent studies showed that analgesic
effects of sinomenine are related to a GABAA receptor and opioid μ-receptor,
both of them are cellular targets for sinomenine in the CNS. Furthermore, in a
previous study, systemic administration (i.p.) of VGSC blockers attenuates
formalin-induced pain behavior in the second phase45. However, peripherally
administered VGSC blocker such as lidocaine suppresses formalin-induced pain
behavior only in the first phase17. Thus, it seems that sinomenine has peripheral
action, in addition to its central actions previously demonstrated by other groups.
45
The gene c-Fos encodes nuclear protein Fos in response to neuronal
activation and has been used as a marker for neuronal activity42. Following the
noxious stimulation, primary afferent nociceptive neurons (Aδ and C-fiber)
transmit pain signal to second order neurons in the superficial dorsal horn of
spinal cord. Thus, c-Fos are expressed in the superficial dorsal horn of spinal cord
after noxious stimulation46 and the reduction of c-Fos expression is commonly
used to show peripheral analgesic effects43. In this study, I found that sinomenine
(50 mg/kg i.p.) decreased formalin-induced c-Fos expression in the superficial
dorsal horn. This result further supports a peripheral action of sinomenine (Fig.
6).
The activity of primary nociceptive neurons (Aδ and C-fiber) is regulated
by ion channels and is important for pain pathway16. Because pain threshold and
intensity are directly correlated with AP threshold and AP frequency, respectively,
in such afferent, I examined whether sinomenine modulate the excitability of
small-sized DRG neurons. Using patch clamp recordings, I first showed that
sinomenine increased AP threshold and decreased AP firing frequency (Fig. 7).
This strongly suggests that sinomenine has an analgesic effect. Consistent with
an involvement of VGSCs in the generation of AP, sinomenine affected VGSCs.
Furthermore, it is well known that VGSCs contribute to AP overshoot phase47
and, sinomenine inhibited AP overshoot phase (Fig. 8). So, the cause of widening
of the AP width could be the reduction of potassium channel activation due to the
sinomenine-induced decrease in VGSCs activation. In the present study, INa was
decreased by sinomenine in a dose dependent manner. Sinomenine has been
demonstrated to inhibit L-type calcium channel and ASIC channel in cortical
neurons48. In addition, vasodilatory action of sinomenine is involved with
inhibition of calcium channels49. These studies show the possibility of ion
46
channel regulation by sinomenine in various cells. On the other hand, present
study proposes that sinomenine regulate VGSCs in primary sensory neurons,
which can produce peripheral analgesic effect by reducing cellular excitability of
nociceptive neurons.
Sinomenine has pharmacological actions on diverse ion channels at a
wide range of concentrations. Sinomenine inhibits voltage-gated calcium
currents in cultured rat cortical neurons at concentration of 0.05–1 μM48. 10 μM
sinomenine inhibited ATP-activated current in HEK293 cells expressing the
P2X3 receptor50. In this study, I observed that sinomenine was effective at a
concentration range of ~ mM on the cellular excitability and VGSCs in small-
sized DRG neurons. Thus, VGSCs may exhibit less sensitivity to sinomenine
compared to VGCCs and P2X3 channels. Likewise, previous studies showed that
diverse ion channels expressed in sensory neurons are targeted by eugenol with
different sensitivity51-53. Given VGCCs and P2X3 channels are also expressed
in DRG neurons, it remains to be determined whether VGCCs and P2X3 channels
in nociceptive neurons are indeed targeted by sinomenine.
The effective analgesic dose of sinomenine (50 mg/kg i.p.) does not elicit
motor activity impairment. When I investigated the effect of sinomenine on
motor function in rota-rod test, the dose of 50 mg/kg (i.p.) were analgesic without
motor activity impairment (Fig. 10). However, I observed motor dysfunction and
some sedative effect at high dose of sinomenine (75 mg/kg i.p.). Because
systemic administration of sinomenine has a side effect such as sedation, gastric
intestine and kidney damage at high dose, it is important to use sinomenine in
low concentrations. In many analgesics as well as sinomenine, the side effects of
systemic administration are an important issue. Thus, various drug administration
methods have been applied to reduce drug concentration and side effects. For
47
example, patch form of lidocaine was used to reduce systemic adverse effects and
effective in neuropathic patients in clinical practice54. Sinomenine also showed
anti-inflammatory effects when topically administrated via patch and spray
formulations55. In present study, I found that i.pl administration of sinomenine
show analgesic effect at the low concentration that did not show analgesic effects
when administered systemically (20 ~25 mg/kg i.pl. versus 25 mg/kg i.p.), imply
its potential therapeutic use as a local analgesic in the periphery.
Based on these findings, I propose a peripheral analgesic mechanism for
sinomenine other than its well-known anti-inflammatory actions, which is
reduction of cellular excitability via the inhibition of INa in small-sized DRG
neurons. Therefore, sinomenine could be a potential pharmacological therapeutic
agent as local anesthetic and analgesics.
48
CHAPTER 2:
Pharmacopuncture with
Scolopendra subspinipes suppresses
mechanical allodynia in oxaliplatin-
induced neuropathy: Mediation by
spinal alpha2-adrenoceptor
*This chapter includes reproductions from an article published by Seo-Yeon Yoon,
Jeong-Yun Lee, Dae-Hyun Roh, Seog Bae Oh. 2018. Pharmacopuncture With
Scolopendra subspinipes Suppresses Mechanical Allodynia in Oxaliplatin-Induced
Neuropathic Mice and Potentiates Clonidine-induced Anti-allodynia Without
Hypotension or Motor Impairment. JOURNAL OF PAIN, 2018, 19, 10, 1157-1168. doi:
10.1016/j.jpain.2018.04.015
49
ABSTRACT
Pharmacopuncture is a new form of acupuncture therapy that combines
acupuncture with herbal medicine by injection of the herbal extract into the
acupoint and has been reported to be more effective than acupuncture alone.
Scolopendra subspinipes (SS) has been used in traditional medicine to treat
chronic neuronal diseases. In this study, I examined the effect of
pharmacopuncture with Scolopendra subspinipes (SSP) into the ST36 acupoint
by using formalin-induced acute inflammatory pain model and oxaliplatin-
induced neuropathic pain model. Subcutaneous (s.c.) treatment with SSP (0.5 %,
20 μl, ST36) did not affect formalin-induced paw licking behavior in both the
first and second phase. On the other hand, SSP (0.5 %, 20 μl, ST36, s.c.)
significantly decreased oxaliplatin-induced mechanical allodynia, which was
completely prevented by acupoint pretreatment of lidocaine. SS injection into a
non-acupoint site had no effect. Intrathecal (i.t.) treatment with yohimbine (25
μg, 5 μl), an alpha2-adrenoceptor antagonist, prevented the anti-allodynic effect
of SSP. In contrast, SSP treatment still produced significant anti-allodynic effect
after treatment with naloxone (20 μg, 5 μl, i.t.), an opioid receptor antagonist, or
methysergide (20 μg, 5 μl, i.t.), a serotonin receptor antagonist. Collectively,
these findings demonstrate that SSP produces an analgesic effect in oxaliplatin-
induced neuropathic pain via neuronal conduction at the acupoint and activation
of spinal alpha2-adrenoceptors.
50
INTRODUCTION
For relieving pain, pharmacological approaches are not the only option.
While medications are targeting the somatic sensation of the pain, non-
pharmacological therapies aim to improve mood states, the affective component
of pain, by modulating the endogenous pain control system9. Acupuncture has
been known to have a great therapeutic potential for relieving pain as a non-
pharmacological therapy56. Especially, Zusanli acupoint (ST36) located below
the knee, on the tibialis anterior muscle has been widely used to elucidate the
analgesic mechanism of acupuncture in animal experiments. Electrical
stimulation at acupoint (EA) is a suitable method to study the analgesic
mechanism of acupuncture even in anesthetized animals57. However, EA has
limitations of short duration and is difficult to apply in non-anesthetized animals.
Pharmacopuncture is a new form of acupuncture therapy that combines
acupuncture with herbal medicine related to the diseases, which is known as a
method of safely stimulating acupoint for a long time in non-anesthetized animals.
Scolopendra subspinipes (SS), a venomous arthropod, has been used in
China and Korea for several hundred years to treat neuronal disorders including
stroke, epilepsy, tetanus, joint problems, neoplasm, and Alzheimer’s disease58,59.
It has been reported that crude extract of SS venom glands is composed of many
ingredients, including histamine, 5-hydroxytryptamine, lipids, polysaccharides,
enzymes, and amino acids 60. Recently, peptides isolated from SS venom were
shown to produce an analgesic effect in animal pain models61,62. However, little
is known about the effect of pharmacopuncture with SS (SSP) on pain. In chapter
2, I examined the effect of SSP into ST36 on animal pain model and further
focused its underlying mechanisms.
51
MATERIALS AND METHODS
Animals
All experimental procedures were reviewed and approved by the
Institutional Animal Care and Use Committee (IACUC) at Seoul National
University. Male C57BL/6 mice weighing 20-28 g and adult male 129S4/SvJae
x C57BL/6J F1 hybrid mice (25- 30 g) were used for the experiment. They were
housed in a temperature- and light-controlled room (22°C ± 2°C, 12-hour
light/dark cycle with lights on at 07:00). Food and water were available as desired.
All animals were habituated to the mouse house 1 week before the start of the
study.
Pharmacopuncture with SS
SS extract (0.5% in saline solution) was obtained from Korean
Pharmacopuncture Institute (Seoul, Korea), and the following is the description
about the extraction method of SS extract. Briefly, dried centipedes (112 mm x 7
g) were used after the head and legs were removed, and 128.6 g of dried
centipedes were immersed in distilled water (DW, 1 L) and stirred at room
temperature for 3 hours. The crude extract was stored in refrigerator overnight to
precipitate, and the supernatant was collected (1´ DW extraction). The DW
extraction using the precipitate was repeated twice (2´ and 3´ DW extraction). All
supernatants of the 1´, 2´, and 3´ DW extracts were passed twice through a filter
paper (Whatman, 0.8 mm) and concentrated under reduced pressure at 70°C for
3 hours using a rotary vacuum evaporator (EYELA, Tokyo, Japan). The viscous
extract was concentrated to an ethanol series (90%, 80%, and 70%) using a rotary
vacuum evaporator. The extract was filtered through Whatman paper in the order
52
of 8.00, 0.45 and 0.10 mm, and lyophilized using a freeze dryer (IlshinBioBase,
Gyeonggido, Korea) for 200 hours. The final product powder to be tested was
dissolved in saline solution at a concentration of 0.005 g/mL (0.5%; pH 11.7) and
stored at 4°C. Saline solution was used as the vehicle for 0.250% or 0.125% SS.
Pharmacopuncture was performed by subcutaneous (s.c.) injection of 20
μl of SS solution at bilateral ST36 acupoints under isoflurane anesthesia. The
ST36 acupoint of mouse was identified based on the human acupoint landmark
and a mouse anatomical reference. It is located in the lateral stifle joint, 5 mm
lateral to the anterior tubercle of the tibia. Vehicle control mice were injected with
the saline into the ST36. Non-acupoint located on the back and buttocks were
selected. 40 μl of SS was injected into a non-acupoint (arbitrary position on the
back) or 20 μl of SS was injected into bilateral non-acupoints (midway between
coccyx and hip joint).
Formalin-induced acute pain model
Formalin test was performed as previously described in chapter 1. 10
minutes after SSP, 20 μl of 1% formalin was injected subcutaneously into the
plantar surface of the right hind paw. Following formalin injection, the animals
were immediately placed in a test chamber and recorded using a video camera
for a period of 40 minutes.
Oxaliplatin-induced neuropathic pain model
Oxaliplatin was purchased from Tocris Bioscience (Bristol, United
Kingdom). A single dose of oxaliplatin (10 mg/kg) was injected i.p. into the
mouse. Vehicle control mice were administrated with an equivalent volume of 5%
dextrose. Oxaliplatin-induced mechanical allodynia was measured using an
53
ascending series of von Frey filaments (North Coast Medical, Morgan Hill, CA).
Mice were acclimated to an acrylic cylinder (6.5 cm diameter, 17 cm height) on
the metal mesh grid for 30 minutes before mechanical allodynia test. Each
monofilament was applied 6 times in ascending order (range of bending force:
0.008, 0.020, 0.040, 0.070, 0.160, 0.400, and 0.600 g) to the midplantar region of
each mouse hind paw. The force of monofilament that elicited paw withdrawal,
licking, or flinching in 3 of the 6 applications was defined as the mechanical Paw
Withdrawal Threshold (PWT).
Drugs
To examine whether peripheral neuronal activation in the acupoint was
related to the effect of SSP, regional nerve block was performed by injection of
lidocaine (0.5% in 20% ethanol; Sigma, St. Louis, MO, 20 μl, s.c.) into the ST36
acupoint 10 minutes before SSP or saline injection. To evaluate the role of the
major descending inhibitory pathway including adrenergic, opioidergic, and
serotonergic neurotransmitter systems in SSP-induced analgesia, yohimbine
(alpha2-adrenoceptor antagonist, 20 μg, 5 μl), naloxone (opioid receptor
antagonist, 20 μg, 5 μl), or methysergide (serotonin receptor antagonist, 20 μg, 5
μl) was administered 5 minutes before SS injection. Using a 50 μl Hamilton
syringe, intrathecal (i.t.) injections were performed by insertion of a 30 G needle
(0.5inch length) into the subarachnoidal space between lumbar vertebrae L5 and
L6. A reflexive flick of the tail indicated a successful entry of the needle in the
subarachnoidal space. All drugs were purchased from Sigma and diluted in saline
except yohimbine, which was diluted in DW.
Statistical Analysis
54
All of the data are presented as mean ± SEM. Statistical analysis was
performed using graphpad prism version 5.1 (Graphpad software). Comparison
between two groups was made using the unpaired student's t test to determine
formalin test. For multiple comparisons, data were analyzed using the one-way
ANOVA followed by Tukey’s test to determine formalin test. Data were analyzed
using two-way ANOVA followed by the Tukey post hoc test for multiple
comparison in mechanical allodynia. Differences with p < 0.05 were considered
significant.
55
RESULTS
SSP do not affect formalin-induced acute inflammatory pain behavior
To investigate the effect of SSP on acute inflammatory pain, I measured
spontaneous pain behavior in the formalin-induced acute inflammatory pain
model (Fig. 11). 1% formalin was injected in right hind paw 10 minutes after
treatment of SSP (0.5%, 20 μl, ST36). Formalin-induced spontaneous pain
behavior was not affected by unilateral (ipsi-ST36) treatment of SSP, while
pharmacopuncture with bee venom (BVP) suppressed formalin-induced
spontaneous pain with a significant analgesic effect in the second phase (Fig.
11A). Again, bilateral treatment of SSP also had no significant effect in formalin-
induced spontaneous pain behavior (Fig. 11B).
SSP suppress Oxaliplatin-induced mechanical allodynia
I investigated whether SSP affected the oxaliplatin-induced neuropathic
pain (Fig. 12). Animals underwent oxaliplatin injection (10 mg/kg i.p.) showed a
significant decrease in mechanical PWT in both hind limbs at 14 days after the
injection (POST) compared with the mean values before the injection (PRE),
indicating induction of mechanical allodynia by oxaliplatin chemotherapy (Fig.
12A and B). Treatment of SSP (20 μl, ST36) in oxaliplatin-treated mice produced
a significant dose-dependent increase in PWT at 30 minutes and 1 hour after SS
injection compared with injection of vehicle (0.9% saline) (Fig. 12B). In contrast,
injection of vehicle (5% dextrose i.p.), instead of oxaliplatin, did not show the
change in PWT at 14 days after injection (Fig. 12C). SSP also had no effect on
PWT in those control mice not treated with oxaliplatin (Fig. 12C). These results
indicate that SS venom extract (0.5%, but not lower concentrations) injected into
56
ST36 location temporarily decreased allodynia (as measured by PWT), and the
effect is over within 2 hours after injection.
Peripheral nerve activation of the specific acupoint is essential for the
analgesic effect of SSP in oxaliplatin-induced mechanical allodynia
Next, I tested whether peripheral nerve activation in the acupoint region
was involved in the SSP-induced anti-allodynic effect. The effect of SSP was
blocked by pretreatment with lidocaine (0.5%, 20 μl) into the ST36 acupoint
(lidocaine + SSP) compared with pretreatment with vehicle only (20% ethanol,
20 μl, vehicle + SSP) (Fig. 13A). Lidocaine without SSP (lidocaine + saline) had
no effect on oxaliplatin-induced mechanical allodynia (Fig. 13A). Furthermore,
I compared effects between acupoint and non-acupoint stimulation (Fig. 13B).
Based on previous studies, the locations of non-acupoint were selected on back
or buttocks. SS injection into non-acupoint sites had no effect on oxaliplatin-
induced decrease in PWT (Fig. 13B).
Spinal alpha2-adrenoceptors mediate the analgesic effect of SSP
I then examined the involvement of the descending endogenous pain
inhibitory system associated with spinal alpha2-adrenergic, opioidergic, and/or
serotonergic system using an i.t. injection of an antagonist of each receptor before
SSP treatment. Yohimbine (alpha2-adrenoceptor antagonist) significantly
decreased the SSP-induced anti-allodynic effect (Fig. 14A), suggesting that SSP-
induced analgesia is mediated by spinal alpha2-adrenoceptors. In contrast,
pretreatment with naloxone (opioid receptor antagonist) or methysergide
(serotonin receptor antagonist) did not affect the SSP anti-allodynic effect (Fig.
14B).
57
Figure 11.
58
Figure 11. Effect of Scolopendra subspinipes
pharmacopuncture (SSP) on formalin-induced acute
inflammatory pain model
(A) Effects of SSP (unilateral injection) on the time course curves of formalin-
induced pain behavior (a). Formalin-induced pain behavior was divided into two
phases and analyzed (b). The bar graph represented the first phase (0-10 min) and
second phase (10-40 min) of formalin-induced pain behavior. Unilateral injection
of SSP did not affect formalin-induced pain behavior in both the first phase and
second phase. Unilateral injection of bee venom pharmacopuncture (BVP)
inhibited formalin-induced pain behavior only in the second phase. *p<0.05,
(one-way ANOVA followed by Tukey’s test) (B) Effects of SSP (bilateral
injection) on the time course curves of formalin-induced pain behavior (a).
Formalin-induced pain behavior was divided into two phases and analyzed (b).
Bilateral injection of SSP did not affect formalin-induced pain behavior in both
first phase and second phase. (unpaired t test, two-tailed)
59
Figure 12.
60
Figure 12. Effect of Scolopendra subspinipes
pharmacopuncture (SSP) on oxaliplatin (OXA)-induced
neuropathic pain model
(A) The experimental design is shown in the time line diagram (B) Intraperitoneal
(i.p.) treatment with OXA (10 mg/kg) showed a significant decrease in
mechanical paw withdrawal threshold (PWT) in both hind limbs after 14 days
(POST) compared with the mean PWT before OXA injection (PRE). SSP
injection into bilateral ST36 locations (0.5%, 0.25%, or 0.125%, 20 μl) dose-
dependently reversed OXA-induced mechanical allodynia. (C) Treatment of
vehicle (5% dextrose) instead of OXA did not affect PWT, and injection of SS or
saline into bilateral ST36 acupoints also did not affect PWT in those vehicle-
treated mice (C). ***P < 0.001, *p<0.05 (two-way ANOVA followed by Tukey’s
test)
61
Figure 13.
62
Figure 13. Peripheral nerve activation of the specific
acupoint is associated with Scolopendra subspinipes
pharmacopuncture (SSP)-induced anti-allodynia
(A) Effect of subcutaneous lidocaine pretreatment on pharmacopuncture with
Scolopendra subspinipes (SSP)-induced anti-allodynic effect in oxaliplatin
(OXA)-treated mice. By comparison with subcutaneous (s.c.) injection of the
vehicle (20% ethanol) with SSP (vehicle + SSP), which induced anti-allodynia,
lidocaine (0.5%/20 μl, s.c.) pretreatment into the ST36 acupoint (lidocaine + SSP)
10 minutes before SSP reversed SSP-induced anti-allodynia. Lidocaine alone
(lidocaine + saline) did not affect OXA-induced mechanical allodynia (vehicle +
saline). (B) SSP reversed oxaliplatin-induced mechanical allodynia with the
bilateral ST36 acupoint injection but did not affect PWT when SSP at non-
acupoint location (back or buttocks). ***p<0.001, *p<0.05 versus vehicle +
saline group (two-way ANOVA followed by Tukey’s test)
63
Figure 14.
64
Figure 14. Effects of pretreatment of intrathecal
injection of yohimbine, naloxone, or methysergide on
Scolopendra subspinipes pharmacopuncture (SSP)-
induced anti-allodynia
Intrathecal treatment of yohimbine (an alpha2-adrenoceptor antagonist),
naloxone (an opioid receptor antagonist) or methysergide (a serotonin receptor
antagonist) was performed 5 minutes before SSP. (A) Yohimbine (25 μg, 5 μl),
prevented the anti-allodynic effect of SSP. (B) In contrast, SSP treatment still
significantly produced anti-allodynic effect after treatment with naloxone or
methysergide. ***p<0.001 versus vehicle + SSP in each group (two-way ANOVA
followed by Tukey’s test)
65
DISCUSSION
In chapter 2, I found out that SSP into bilateral ST36 acupoint in adult
mice relieved mechanical allodynia associated with oxaliplatin chemotherapy-
induced neuropathy. The SSP-induced anti-allodynia was mediated via peripheral
neuronal activation at the SSP acupoint and via activation of spinal alpha2-
adrenoceptors.
Compared to conventional acupuncture combined with oral medication,
the benefits of pharmacopuncture are synergistic effects of acupuncture and
pharmacological medicine. Pharmacopuncture has more immediate effects
compared to oral drug administration because the pharmacological materials are
absorbed directly, without passing through the gastrointestinal tract. In non
acupoint experiment, a total 40 μl of SS, the same amount with analgesic effect
when treated into ST36 (20 μl on each ST36 acupoint), was injected into an
arbitrary non-acupoint location but there was no effect (Fig. 13B). Therefore, the
analgesic effect of SSP may not be due to the systemic effect of SS.
Since peptides isolated from SS venom produce an analgesic effect by
inhibiting TRPV1 and Nav1.761,62, there is a possibility that the analgesic effect
of SS injection into ST36 could be mediated by inhibiting TRPV1 and Nav1.7 at
ST36. However, lidocaine into ST36 point itself did not affect the pain sensation
in both oxaliplatin and vehicle-treated mice (Fig. 13A). Furthermore,
anatomically, ST36 is quite close to the deep peroneal nerve and the tested plantar
surface of the paw in this study is mainly innervated by the tibial nerve (partially
the sural and saphenous nerves) rather than peroneal nerve63. Therefore, it is
plausible that the analgesia induced by SSP at ST36 is not just mediated by the
simple pharmacologic effect of nerve agents especially into a peroneal nerve.
66
Peripheral nerve block by pretreatment of lidocaine at ST36 completely
reversed the SSP-induced analgesia (Fig. 13A). These findings indicate that the
analgesic effect of SSP on oxaliplatin-induced neuropathy might be related to
peripheral neuronal activation in the ST36 acupoint. Acupoint stimulation
activate peripheral nociceptive neurons and induce a sensation of heaviness,
soreness or numbness (deqi, a composite of unique sensations), which has been
shown to be essential for the analgesic effects of acupuncture56 and induces
neuronal signals that transmit to the spinal cord and then to the brain, resulting in
activation of the descending endogenous pain inhibitory system57. With this in
mind, I evaluated whether the anti-allodynic effect of SSP is mediated by the
descending endogenous pain inhibitory system associated with spinal alpha2-
adrenergic, opioidergic, and/or serotonergic system. I found that intrathecal
pretreatment with yohimbine blocked the SSP-induced anti-allodynia in
oxaliplatin-induced neuropathic mice, whereas pretreatment with naloxone or
methysergide did not (Fig. 14). This result indicates that spinal alpha2-
adrenoceptors, but not opioid or serotonin receptors, are involved in the analgesic
effect of SSP.
SSP-induced neuronal signaling might activate the descending
noradrenergic system and spinal a2-adrenoceptors, leading to the analgesic effect
on oxaliplatin chemotherapy-induced neuropathic pain. There are several articles
demonstrating that the physiological mechanisms underlying pharmacopuncture
with bee venom, which is another useful venom for oriental medicine, induced
anti-nociceptive and anti-inflammatory effect in various rodent models27,64. BVP
into the ST36 acupoint potently activates catecholaminergic neurons in locus
coeruleus, ultimately leading to the activation of descending noradrenergic
pathway into the spinal cord65. Moreover, this increased activity of spinal
67
noradrenergic neurons mediates an alpha2-adrenoceptor, which is mainly
involved in pharmacopuncture-induced effect in acute and neuropathic pain
models. However, it is unclear how the SSP-induced activation of descending
noradrenergic pathway leads to significant analgesic effects in chemotherapy-
induced neuropathic pain. Further studies need to be performed to delineate these
concerns.
This study demonstrates that SSP suppresses oxaliplatin chemotherapy-
induced mechanical allodynia via peripheral nerve activation in the injected
acupoint and activation of spinal alpha2-adrenoceptors. Although this study
shows a potential therapeutic effect of SSP in established oxaliplatin-induced
pain, the prevention of chemotherapy-induced pain is also important clinically.
Further study is needed to evaluate the effect of SSP on the development of
chemotherapy-induced neuropathic pain.
68
CHAPTER 3:
Both acute fasting and refeeding
alleviate inflammatory pain but via
different pathways
*This chapter has been largely reproduced from articles published by
Jeong-Yun Lee, Grace J Lee, Pa Reum Lee, Chan Hee Won, Doyun Kim, Youngnam Kang,
Seog Bae Oh, The analgesic effect of refeeding on acute and chronic inflammatory pain.
Scientific Reports, 2019, 14;9(1):16873. doi: 10.1038/s41598-019-53149-7
69
ABSTRACT
Pain is susceptible to various cognitive factors. Suppression of pain by
hunger is well known, but the effect of food intake after fasting (i.e. refeeding)
on pain remains unknown. In the present study, I examined whether inflammatory
pain behavior is affected by 24h fasting and 2h refeeding. In formalin-induced
acute inflammatory pain model, fasting suppressed pain behavior only in the
second phase and the analgesic effect was also observed after refeeding.
Refeeding with non-calorie agar produced an additional analgesic effect
compared with fasting. Besides, administration of glucose (i.p.) after fasting,
which mimics calorie recovery following refeeding, induced a greater analgesic
effect than vehicle group. Administration of opioid receptor antagonist (naloxone,
i.p.) and cannabinoid receptor antagonist (SR 141716, i.p.) reversed fasting-
induced analgesia, but did not affect refeeding-induced analgesia in acute
inflammatory pain model. Taken together, these results show that fasting and
refeeding produce analgesia through different mechanisms, suggesting that
endocannabinoid and opioid system is only involved in fasting-induced analgesia,
whereas refeeding-induced analgesia is associated with eating behavior and
calorie effect.
70
INTRODUCTION
Pain perception is a multifaceted experience, largely divided into sensory
and affective dimension1,66. The sensory dimension of pain provides sensory-
discriminative information such as location, quality, and intensity. Affective pain
consists of hedonic aspect (pleasantness/unpleasantness) and affective-
motivation aspect which creates behavior to escape from pain as a secondary pain
effect67. Since pain is both a sensation and the emotion which is an unpleasant
state motivating an organism to react in favor of its survival, it is susceptible to
modulation by cognitive factors3,68.
Hunger is well known as a powerful driving force to change cognition.
Several clinical evidences have shown that mood states, such as confusion, anger
and tension can be improved by limiting food intake69. Furthermore, therapeutic
fasting is also effective for pain relief and mood enhancement regardless of body
weight change in chronic pain patient70,71. In various animal pain models, it is
also well established that fasting or calorie restriction also has an analgesic
effect72-75. Interestingly, recent studies have revealed that a part of the
parabrachial nucleus (PBN) is involved in the suppression of pain response by
fasting76,77. Endogenous opioid and endocannabinoid system not only play a
critical role in both homeostatic and hedonic aspects of feeding but also are
involved in the endogenous pain inhibitory system78,79. Therefore, activation of
opioid and endocannabinoid system after fasting is likely to modulate pain.
However, the relationship between the analgesic effect after fasting and these
endogenous pain control systems have not been elucidated.
While several studies have found the fasting-induced analgesia, little is
known about the effect of food intake after fasting on pain. Previous study has
71
revealed that hedonic drinking induces analgesic effect in fasted animal80.
Moreover, food ingestion after fasting reduced pain perception in healthy
volunteers81. In animal study, thermal pain threshold also increased at returning
to free feeding after calorie-restriction82. Thus these studies strongly suggest the
possibility that food intake after fasting can suppress pain. Nevertheless, the ph
enomenon or the mechanism for it is still unknown.
In this study, I thus sought to explore the change of pain behavior
following fasting and refeeding using inflammatory pain conditions with the
formalin-induced acute pain model. I found that fasting-induced analgesia was
mediated by opioid and endocannabinoid system. On the other hand, refeeding-
induced analgesia is mediated by eating behavior and calorie recovery.
72
MATERIALS AND METHODS
Animals
Male C57BL/6 mice weighing 18-25 g were purchased from DooYeol
Biotech (Korea) and maintained with standard lab chow (pellet diet) and water
ad libitum except when food was removed for deprivation experiments. All
experimental procedures were reviewed and approved by the Institutional Animal
Care and Use Committee (IACUC) at Seoul National University (SNU-170705-
1, SNU-180518-1). All experiments were performed in accordance with relevant
guidelines and regulations that was confirmed by IACUC.
Formalin-induced pain model
Formalin test was performed as previously described in chapter 1. The
time mice spent licking was measured during each 5 minutes by an observer who
was blinded to the treatment. Formalin-induced licking behaviors during 0-10min
after formalin injection represented the first phase and during 10-40min after
formalin injection represented the second phase. The total sum of the licking
times for each phase was statistically analyzed.
von Frey test in naïve mice
To assess mechanically evoked pain, both the 50% paw withdrawal
threshold and paw withdrawal frequency was measured using von Frey filaments
(North Coast Medical, Morgan Hill, CA, USA). Mice were acclimated in cage at
least for a week and then adapted in an acrylic cylinder (6.5 cm diameter, 17 cm
height) on the metal mesh floor before the experiment at least three times. All
animals left to an acrylic cylinder on the metal mesh floor for 30min-1h prior to
73
mechanical test. The 50% paw withdrawal threshold was determined based on
the up-down method with an ascending series of von Frey filaments (0.16 g, 0.4
g, 0.6 g, 1 g, and 2 g). The response frequency was used to assess the paw
withdrawal frequency to the repeated application of a single von Frey filament
(0.16 g or 0.6 g, 10 times each).
Hargreaves test in naïve mice
To assess heat-evoked thermal pain, paw withdrawal latency was
measured using Hargreaves method (IITC Life Science Plantar Test, Victory Blvd
Woodland Hills, CA, USA) with a glass surface, heated to 30 °C. Thermal latency
was measured 4 to 7 times and averaged.
Administration of drug
Naloxone (Tocris) was diluted in 0.9% saline. SR 141716 (Tocris) was
diluted in 0.9% saline with 10% DMSO and 1% tween 80. These drugs were
intraperitoneally (i.p.) injected at a dose of 10 mg/kg in a volume of 10 ml/kg
body weight.
Feeding schedule for pain behavior test
Food deprivation began between the hours of 09.00-13.00. For refeeding
with the non-calorie agar pellet experiment, additive-free agar powder was
melted in tap water (4%, in a microwave oven and then cooled in a refrigerator)
and cut into about 1.5 x 1 x 0.5 cm. After 24h food deprivation, mice had free
access to 4% agar for 2h. Water was freely accessible in all experiments.
74
Measurement of body weight, blood glucose level and food intake
Glucose levels were detected in blood samples collected from the tail
vein using an ACURA PLUS (automated glucometer, Korea). Body weight was
measured before and after 24h food deprivation and 2h and 24 h of refeeding.
And then, fasting and refeeding body weight was normalized to body weight
before fasting. The weight of the diet was measured per cage by weighing food
before and after ingestion and divided by the number of mice per cage.
Administration of D-glucose
D-glucose was diluted in 0.9% saline and injected at a dose of 1 mg/kg
(i.p.) in a volume of 10 ml/kg body weight. In the formalin test, after 15min of
D-glucose injection, formalin was injected in 24h fasted mice.
Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 5.0
(GraphPad Software, USA). Comparison between two groups was made using
the unpaired or paired Student's t-test. For multiple comparisons, data were
analyzed using the one-way ANOVA, repeated ANOVA followed by the post hoc
Bonferroni test. Detailed statistics for each experiment were shown in the figure
legend. Data are presented as mean ± SEM. Differences with p<0.05 were
considered significant.
75
RESULTS
Both fasting and refeeding suppress acute inflammatory pain behavior
To investigate the effect of fasting and refeeding on acute pain behavior,
I measured spontaneous pain behavior in the formalin-induced acute
inflammatory pain model and mechanical/thermal pain threshold in naïve mice
(Fig. 15). Consistent with previous studies74,77,83, 24h acute fasting suppressed
formalin-induced spontaneous pain behavior with a significant analgesic effect
only in the second phase (Fig. 15A). As compared with the free fed group, 2h
refed group had a significant analgesic effect only in the second phase, and this
analgesic effect was disappeared at 24h refeeding (Fig. 15A). In naïve mice, both
paw withdrawal threshold for mechanical stimuli and paw withdrawal latency for
thermal stimuli were not affected by 24h fasting and remained unchanged after
2h/24h refeeding (Fig. 15B).
To make satiety condition, mice were fasted for 24h and then allowed to
free access to normal chow for 2h. Induction of satiety was confirmed by body
weight, blood glucose level, food intake and stomach size (Fig. 16). After 24h
fasting, body weight and blood glucose level decreased, which was recovered
after 2h refeeding (Fig. 16 A and B). 2h refeeding increased food intake and
stomach was significantly expanded compared to the free fed mice (Fig. 16C and
D).
These results suggest that both fasting and refeeding produces an
analgesic effect in acute inflammatory pain but did not affect non-inflammatory
pain (nociception).
76
Refeeding of non-calorie agar pellet induces analgesic effect
Next, I investigated whether eating behavior or calorie recovery is
involved in refeeding-induced analgesia. To eliminate the effect of calorie
recovery, I used non-calorie agar pellet.
In the formalin-induced acute pain model, both refeeding of non-calorie
agar and normal lab chow had an analgesic effect only in the second phase (Fig.
17A and B). Interestingly, the agar-refeeding group had a greater analgesic effect
than fasting or normal chow-refeeding group (Fig. 17C), whereas there was no
difference in the analgesic effect between high-calorie chow (Oreo) and normal
chow (Fig. 17C). As compared to refeeding with normal chow, refeeding of non-
calorie agar for 2h did not increase stomach size, body weight, and blood glucose
level (Fig. 17D). Thus, agar-refeeding engaged eating behavior but did not induce
satiety signal by calorie recovery or stomach expansion.
These results indicate that eating behavior during refeeding contributes to
refeeding-induced analgesia, regardless of calorie.
Intraperitoneal administration of glucose induces analgesic effect
An additional analgesic effect by refeeding with non-calorie agar
suggests that satiety signal that consists of calorie recovery and stomach
expansion may have interfered with refeeding-induced analgesia. So I next
investigated the effect of calorie recovery on refeeding-induced analgesia. To
determine the effect of calorie recovery on pain without engaging eating behavior
and stomach expansion, I injected D-glucose (1 g/kg, i.p.).
Formalin was injected 15min after D-glucose injection in fasted mice (Fig.
18Aa). Compared with the vehicle (0.9% saline, i.p.) treated group, D-glucose
treated group had no significant difference in the sum of formalin-induced
77
spontaneous pain behavior from 10min to 40min (Fig. 18Ab and c). However,
only when comparing formalin-induced pain behavior from 10min to 30min, pain
behavior was significantly decreased in D-glucose treated group (Fig. 18Ab and
d). As compared to the vehicle group, administration of D-glucose increased
blood glucose level over time, peaked after 15 minutes and recovered after 1h
(Fig. 18B).
Taken together, these findings suggest that calorie recovery after fasting
may serve as an additional factor for refeeding-induced analgesic effect.
Opioid and endocannabinoid system contribute to fasting-induced analgesia,
but not refeeding-induced analgesia
To determine the involvement of opioid and endocannabinoids system in
fasting and refeeding-induced analgesia, I used naloxone (opioid receptor
antagonist) and SR 141716 (cannabinoid receptor (CB1) antagonist). In the
previous study, it was confirmed that SR 141716 significantly inhibit food
intake after fasting at dose of 10 mg/kg but not at 3 mg/kg84,85. I also confirmed
that 3mg/kg of naloxone (i.p.) had no effect on fasting-induced analgesia (data
not shown). Naloxone (10 mg/kg, i.p.) and SR 141716 (10 mg/kg, i.p.) was
administrated 30min before formalin injection (Fig. 19A). In formalin-induced
acute inflammatory pain model, both naloxone and SR 141716 inhibited fasting-
induced analgesia but did not affect refeeding-induced analgesia (Fig. 19B-E).
These results suggest that endogenous opioid and endocannabinoid
system mediate fasting-induced analgesia, but not refeeding-induced analgesia.
Refeeding might recruit distinctive analgesic factors from fasting-induced
analgesia.
78
Figure 15.
79
Figure 15. The effect of fasting and refeeding on acute
pain behavior
(A) Experimental design and schedule for formalin test (a). Time course of
spontaneous pain behavior following intraplantar injection of formalin (b).
Formalin-induced pain behavior was divided into two phases and the total sum
of the licking times for each phase was statistically analyzed; Free fed (n=8),
Fasted (n=9), 2h refed (n=8), 24h refed (n=7) (c). (B) The effect of 24h fasting
and 2h refeeding on paw withdrawal threshold and frequency for mechanical
stimuli (n=6) and paw withdrawal latency for thermal stimuli (n=5). Data are
presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (A one-way ANOVA
followed by Bonferroni test, B repeated measures ANOVA)
80
Figure 16.
81
Figure 16. Changes in body weight, blood glucose level
and food intake by refeeding in naïve mice
(A, B) Change in body weight and blood glucose level after refeeding in naïve
mice. Refeeding for 2h after 24h fasting caused weight gain; (n=5). The decrease
in blood glucose level due to fasting was also increased after 2h refeeding; (n=7).
(C) Stomach expansion induced by refeeding and food inside stomach (D)
Changes in food intake after refeeding. Compared with free fed group, the
amount of food intake for 2h was significantly increased in 24h fasted group. The
difference in food intake between two groups decreased after 2h refeeding. Data
are presented as mean ± SEM. *p,0.05, ***p<0.001 (unpaired t test, two-tailed)
82
Figure 17.
83
Figure 17. The analgesic effect of refeeding with non-
calorie agar on inflammatory pain behavior
(A, B) Effect of 2h refeeding non-calorie agar or normal chow on formalin-
induced pain behavior; Free fed (n=9), Agar-refed (n=12), Free fed (n=12),
Normal chow-refed (n=12). (C) Comparison of 2nd phase according to
experimental groups; Free fed (n=19), Fasted (n=12), Agar-refed (n=12), Normal
chow-refed (n=12), Oreo-refed (n=4). (D) Change in stomach size, body weight,
blood glucose level and food intake after agar refeeding in naïve mice. Data are
presented as mean ± SEM. **p<0.01, ***p<0.001 (A, B unpaired t-test, two-
tailed, C one-way ANOVA followed by Bonferroni test)
84
Figure 18.
85
Figure 18. The analgesic effect of intraperitoneal
administration of D-glucose on inflammatory pain
behavior
(A) Experimental design and schedule for formalin test (a). Time course of
spontaneous pain behavior following intraplantar injection of formalin after D-
glucose (1g/kg) administration (b); Free fed (n=11), Fasted (n=14), D-glucose
treated (n=8). 2nd phase of formalin test was divided into 10 to 40min and 10 to
30min and analyzed (c, d). (B) Blood glucose level after intraperitoneal
administration of D-glucose (1g/kg); D-glucose treated (n=4), Vehicle treated
(n=3). Data are presented as mean ± SEM. **p<0.01, ***p<0.001 (A one-way
ANOVA followed by Bonferroni test)
86
Figure 19.
87
Figure 19. The effect of opioid receptor and cannabinoid
receptor (CB1) antagonist on fasting and refeeding-
induced analgesia
(A) Experimental design and schedule for formalin test. Naloxone (opioid
receptor antagonist, 10 mg/kg) and SR 141716 (CB1 receptor antagonist, 10
mg/kg) were intraperitoneally (i.p.) administered 30 min before the formalin
injection. (B, C) Time course of spontaneous pain behavior following intraplantar
injection of formalin; Free fed-vehicle (n=9), Free fed-naloxone (n=7), 24h
fasted-vehicle (n=13), 24h fasted-naloxone (n=11), 2h refed-vehicle (n=12), 2h
refed-naloxone (n=10) / Free fed-vehicle (n=11), Free fed-SR 141716 (n=11),
24h fasted-vehicle (n=9), 24h fasted-SR 141716 (n=9), 2h refed-vehicle (n=10),
2h refed-SR 141716 (n=9). (D, E) Formalin-induced pain behavior was divided
into two phase and the total sum of the licking times for each phase was
statistically analyzed. Data are presented as mean ± SEM. **p<0.01, ***p<0.001
(D, E one-way ANOVA followed by Bonferroni test)
88
DISCUSSION
In chapter 3, I compared pain behaviors between fasting and refeeding
mice and discovered that refeeding only alleviates pathological pain induced by
inflammation. In the formalin-induced acute inflammatory pain model, refeeding
suppressed only spontaneous pain behavior especially in the second phase which
represents inflammatory pain, whereas the first phase of the formalin test, the von
Frey test and Hargreaves test were not affected by both fasting and refeeding. I
confirmed that fasting and refeeding produces analgesic effect through different
mechanisms where fasting produces analgesic effect via the opioid and
endocannabinoid system, but these systems are not involved in refeeding-induced
analgesia. Both refeeding of non-calorie agar and the calorie recovery by D-
glucose injection (i.p.) after fasting had an additional analgesic effect, compared
to fasting-induced analgesia.
Feeding behavior is crucial for maintaining homeostasis and it is well
investigated that pain perception is changed in eating disorder patients86,87. Since
opioid and endocannabinoid system are critical modulators of pain as well as
feeding78,79, these systems are important factors in determining the relationship
between feeding and pain. Diurnal fluctuations in pain sensitivity were not
caused by circadian rhythm but food deprivation, which was related to opioid
system72. Intermittent fasting produced analgesic effect via kappa-opioid system
in spinal cord88. It is also well known that leptin-deficient (ob/ob) obese mice
have increased pain threshold by activating endocannabinoid system in
descending pain pathway89. In the present study, I found that acute fasting leads
to analgesic effects in acute inflammatory pain through opioid and cannabinoid
system, whereas the analgesic effect of refeeding was not associated with these
89
systems (Fig. 19). Thus refeeding might recruit distinctive factors which are
different from fasting-induced analgesia.
Eating behavior and satiety signal such as calorie recovery and stomach
expansion may result from refeeding after fasting. I determined which factors
play critical roles in refeeding-induced analgesia. First, I identified the effect of
eating behavior on pain. Several pre-clinical studies have suggested that pain
signals are suppressed during drinking or eating regardless of calorie80,90,91.
Moreover, previous study also showed that not only drinking a sucrose solution
but also water drinking increased pain thresholds in the Hargreaves test of
thermal sensitivity80. However, these studies are not suitable to investigate the
effects of refeeding on pain because eating and pain signals exist at the same time.
In the present study, I performed a pain behavior test after eating behavior was
completed, and found that even eating non-calorie food produces a greater
analgesic effect than fasting-induced analgesia, indicating that eating behavior
itself can suppress pain perception, regardless of calorie (Fig. 17).
Next, I examined the effect of calorie recovery after fasting on pain.
Glucose administration after fasting is known to enhance neuroplasticity and
cognitive function31,92, so it is possible that pain perception is also affected by
glucose administration92. In the present study, it was found that glucose
administration (i.p.) had a greater analgesic effect compared to fasting only while
blood glucose level remained elevated (Fig. 18), suggesting that refeeding-
induced analgesia is also associated with a calorie recovery. Therefore, refeeding-
induced analgesia is multiple phenomena occurring simultaneously, which are
clearly distinctive from simple fasting-induced analgesia.
Finally, stomach expansion might affect pain perception. Physiologically,
slight distention of the stomach does not cause a significant increase in gastric
90
pressure, but severe stomach distension may activate the affective pain system93.
Hence it is possible that the affective component of pain from significant stomach
expansion may interfere with refeeding-induced analgesia. Furthermore, PBN
receives satiety signal such as ingestion, gut distention and satiety hormones
directly from nucleus of the solitary tract (NTS)94 and is activated by not only
meal-related satiety but also noxious stimuli95. Therefore, fullness due to stomach
expansion could interfere with the analgesic effect of refeeding in formalin test
by activating PBN (Fig. 17C and D).
I have not yet determined the brain circuits that mediate refeeding-
induced analgesia. Painful stimuli, such as formalin or electrical shock, is known
to induce c-Fos (neural activity marker) expression in PBN77,96. A previous study
reveals that hunger suppress inflammatory pain behavior by inhibiting PBN via
the agouti-related protein (AgRP)-expressing neuron77. However, it is well
known that the expression of c-Fos in PBN is increased by 2h refeeding after 20-
40h acute fasting97-99 which is involved in determination of meal size and meal
termination during refeeding100. Hence, I suggest that 2h refeeding-induced
analgesia is less likely mediated by PBN.
Feeding and fasting drive oscillation of energy metabolism. These
oscillations are known to have positive effects not only on healthy lifespan but
also on neural circuits related to cognition and emotion29. Thus, alternation of
fasting-refeeding may have many benefits to modulate pain. Based on these
findings, I propose that both fasting and refeeding modulate pathological pain
more effectively through different mechanisms.
91
CONCLUSION
Pain is a highly individualized phenomenon with the mismatch between
pain complaint and pathological condition. Therefore, integrated multimodal care
is required for pain management. In this thesis, I have explored various
therapeutic approaches to pain based on pain pathways (the lateral pain pathway,
the medial pain pathway, and the descending pain pathway).
In the first chapter, I tried to reduce pain by regulating the lateral pain
pathway. I showed that sinomenine can produce peripheral analgesic effect by
inhibiting INa in nociceptive afferent neurons. In the second chapter, I wanted to
find a form of pain treatment by enhancing the descending pain pathway. I found
that acupoint stimulation with Scolopendra subspinipes activated the descending
pain inhibitory pathway associated with the spinal alpha2-adrenergic system. In
the third chapter, I sought to investigate whether the medial pain pathway could
be affected by modulation of feeding patterns. Both the fasting and refeeding
produced analgesic effect only in pathological pain. The nociception that mainly
related to the lateral pain pathway, was not affected by fasting and refeeding.
This thesis shows that the regulation of each pain pathway has potential
therapeutic effects. However, it cannot be confirmed whether these approaches
control pain in the patients who are not managed by conventional pain treatment.
Thus, further research is needed regarding combination therapy for clinical
applications.
92
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국문초록
통증 조절을 위한 다각적 접근:
국소 진통제, 약침 요법 및 시간 제한 섭식에
관한 연구
통증은 국제 통증 연구 협회 (IASP)에 의해 실질적/잠재적
조직 손상과 관련된 불쾌한 감각 또는 정서적 경험으로 정의된다.
통증은 감각 및 정서적 요소를 모두 가지고 있기 때문에, 기존의
약리학적 접근법 (NSAID 및 오피오이드) 만으로는 통증을
효과적으로 조절하는 것이 어렵다. 따라서 본 학위 논문은 통증
관리에 대한 다각적인 접근 방식을 모색하기 위해 국소 진통제
(sinomenine), 약침 요법 (Scolopendra subspinipes, 오공 약침) 및 시간
제한 섭식 (금식 / 금식 후 재 섭식)의 통증 조절 가능성을 주요 연구
대상으로 하였다.
첫 번째 장에서는 sinomenine이 통각 수용 감각 신경의 흥분을
감소시킴으로써 말초에서 진통 효과를 유도 할 수 있음을 밝혔다.
Sinomenine의 복강 내 투여는 포르말린으로 유도된 통증 행동 및 c-
Fos 발현을 감소 시켰으며, sinomenine이 전압에 따라 개폐 되는 소디
99
움 채널을 억제함을 통각 수용 감각신경에서 확인하였다. 또한
sinomenine을 하지 말단에 국소 투여하였을 때도 통증 행동 감소가 관
찰되었다. 이러한 결과는 sinomenine이 국소 마취제 및 진통제로서 적
용 가능함을 제시한다.
두 번째 장에서는 오공 약침을 이용한 경혈 자극이 척수에 존
재하고 있는 alpha2 아드레날린 수용체를 통해 진통 효과를 유도한
다는 것을 밝혔다. 오공약침을 족삼리 영역에 피하 투여 시 옥살리플
라틴 처치로 인해 유발된 신경 병증성 통증을 감소시킴을 확인하였으
며, alpha2 아드레날린 수용체의 길항제로 알려진 yohimbine을 척수 내
에 주입하였을 때 오공 약침으로 유도된 진통 효과가 억제되었다. 따
라서, 오공 약침에 의한 경혈 자극은 하행성 통증 억제 시스템을 활
성화시켜 진통 효과를 나타내며, 이는 오공 약침을 이용한 통증 관리
의 가능성을 시사한다.
세 번째 장에서는 금식과 재 섭식 시 보이는 진통 효과에
중점을 두었다. 자율 섭식과 비교하였을 때, 24시간 금식과 2 시간 재
섭식은 모두 진통 효과를 보였으나 진통 효과를 유도하는 기전이
다름을 확인하였다. 24 시간 금식 시 유도된 통증 억제 효과는
엔도카나비노이드와 오피오이드 시스템이 관여하는 반면, 2 시간 재
섭식으로 유도된 진통 반응에는 섭식을 하는 행동 자체와 칼로리
회복이 관여 하였다. 따라서 섭식 조절 (시간 제한 섭식)은 통증
관리를 위한 새로운 치료 전략이 될 수 있을 것이라 기대된다.
100
주요어: 통증; sinomenine; 소디움 채널; 오공 약침; alpha2 아드레날린
수용체; 금식; 시간 제한 섭식
학번: 2015-22669