Downregulation of selective microRNAs in trigeminal ganglion neurons following inflammatory muscle pain.
ABSTRACT Active regulation of gene expression in the nervous system plays an important role in the development and/or maintenance of inflammatory pain. MicroRNA (miRNA) negatively regulates gene expression via posttranscriptional or transcriptional inhibition of specific genes. To explore the possible involvement of miRNA in gene regulation during inflammatory pain, we injected complete Freund's adjuvant (CFA) unilaterally into the rat masseter muscle and quantified changes in neuron-specific mature miRNAs in the trigeminal ganglion (TG). Real-time reverse-transcription polymerase chain reaction revealed significant, but differential, downregulation of mature miR-10a, -29a, -98, -99a, -124a, -134, and -183 in the ipsilateral mandibular division (V3) of the TG within 4 hr after CFA. In contrast, levels of tested miRNAs did not change significantly in the contralateral V3 or the ipsilateral ophthalmic and maxillary divisions of the TG from inflamed rats, nor in the ipsilateral V3 of saline-injected animals. The downregulated miRNAs recovered differentially to a level equal to or higher than that in naive animals. Full recovery time varied with miRNA species but was at least 4 days. Expression and downregulation of some miRNAs were further confirmed by in situ hybridization of TG neurons that innervate the inflamed muscle. Although neurons of all sizes expressed these miRNAs, their signals varied between neurons. Our results indicate that miRNA species specific to neurons are quickly regulated following inflammatory muscle pain.
Article: Basic science of pain.[show abstract] [hide abstract]
ABSTRACT: The origin of the theory that the transmission of pain is through a single channel from the skin to the brain can be traced to the philosopher and scientist René Descartes. This simplified scheme of the reflex was the beginning of the development of the modern doctrine of reflexes. Unfortunately, Descartes' reflex theory directed both the study and treatment of pain for more than 330 years. It is still described in physiology and neuroscience textbooks as fact rather than theory. The gate control theory proposed by Melzack and Wall in 1965 rejuvenated the field of pain study and led to further investigation into the phenomena of spinal sensitization and central nervous system plasticity, which are the potential pathophysiologic correlates of chronic pain. The processing of pain takes place in an integrated matrix throughout the neuroaxis and occurs on at least three levels-at peripheral, spinal, and supraspinal sites. Basic strategies of pain control monopolize on this concept of integration by attenuation or blockade of pain through intervention at the periphery, by activation of inhibitory processes that gate pain at the spinal cord and brain, and by interference with the perception of pain. This article discusses each level of pain modulation and reviews the mechanisms of action of opioids and potential new analgesics. A brief description of animal models frames a discussion about recent advances regarding the role of glial cells and central nervous system neuroimmune activation and innate immunity in the etiology of chronic pain states. Future investigation into the discovery and development of novel, nonopioid drug therapy may provide needed options for the millions of patients who suffer from chronic pain syndromes, including syndromes in which the pain originates from peripheral nerve, nerve root, spinal cord, bone, muscle, and disc.The Journal of Bone and Joint Surgery 05/2006; 88 Suppl 2:58-62. · 3.23 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Inflammatory pain manifests as spontaneous pain and pain hypersensitivity. Spontaneous pain reflects direct activation of specific receptors on nociceptor terminals by inflammatory mediators. Pain hypersensitivity is the consequence of early posttranslational changes, both in the peripheral terminals of the nociceptor and in dorsal horn neurons, as well as later transcription-dependent changes in effector genes, again in primary sensory and dorsal horn neurons. This inflammatory neuroplasticity is the consequence of a combination of activity-dependent changes in the neurons and specific signal molecules initiating particular signal-transduction pathways. These pathways phosphorylate membrane proteins, changing their function, and activate transcription factors, altering gene expression. Two distinct aspects of sensory neuron function are changed as a result of these processes, basal sensitivity, or the capacity of peripheral stimuli to evoke pain, and stimulus-evoked hypersensitivity, the capacity of certain inputs to generate prolonged alterations in the sensitivity of the system. Posttranslational changes largely alter basal sensitivity. Transcriptional changes both potentiate the system and alter neuronal phenotype. Potentiation occurs as a result of the up-regulation in the dorsal root ganglion of centrally acting neuromodulators and simultaneously in the dorsal horn of their receptors. This means that the response to subsequent inputs is augmented, particularly those that induce stimulus-induced hypersensitivity. Alterations in phenotype includes the acquisition by A fibers of neurochemical features typical of C fibers, enabling these fibers to induce stimulus-evoked hypersensitivity, something only C fiber inputs normally can do. Elucidation of the molecular mechanisms responsible provides new opportunities for therapeutic approaches to managing inflammatory pain.Proceedings of the National Academy of Sciences 08/1999; 96(14):7723-30. · 9.74 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Recent data support an important role for calcitonin gene-related peptide (CGRP) in deep tissue nociceptive processing. Using real-time reverse transcriptase polymerase chain reaction (RT-PCR), radioimmunoassay, immunohistochemistry and behavioral testing, we studied the early time course of CGRP mRNA and protein expression as well as nociceptive behavior following muscle inflammation. A rapid and significant increase in CGRP mRNA occurred in the mandibular division (V3) of the ipsilateral trigeminal ganglion at 30 minutes, 4 and 24 h after the injection of complete Freund's adjuvant as an inflammatory agent into rat masseter muscle. No change in mRNA occurred in the ipsilateral ophthalmic and maxillary divisions (V1/V2) or in the contralateral V3. The levels of immunoreactive calcitonin gene-related peptide (iCGRP) in the ipsilateral V3 significantly increased at 1, 4 and 24 h following muscle inflammation. In contrast, no change occurred in iCGRP levels in either the ipsilateral V1/V2 or contralateral V3. When saline was injected into the masseter muscle, the levels of mRNA or iCGRP did not change in the ipsilateral V3 suggesting that the biochemical changes are specific to CFA-induced muscle inflammation. The number of muscle afferent neurons immunoreactive for CGRP was significantly reduced compared with control at 1, 4 and 24 h in the ipsilateral but not in the contralateral trigeminal ganglion following inflammation. This decrease in the ipsilateral ganglion may indicate a loss of intrasomatic CGRP as a result of increased axonal transport away from the neuronal cell body and/or release of CGRP. Behavioral testing showed a reduction in head withdrawal thresholds bilaterally from 30 min through 24 h following muscle inflammation. Thus upregulation of CGRP mRNA and iCGRP levels are temporally related to the development of inflammation and lowered pain thresholds. The present data support the hypothesis that CGRP is upregulated during deep tissue inflammation and suggest that gene transcription is involved in this upregulation.Neuroscience 01/2007; 143(3):875-84. · 3.12 Impact Factor
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Downregulation of selective microRNAs in trigeminal ganglion
neurons following inflammatory muscle pain
Guang Bai*, Rajini Ambalavanar, Dong Wei and Dean Dessem
Address: Department of Biomedical Sciences, Program in Neuroscience, University Maryland Dental School, Baltimore, MD, USA
Email: Guang Bai* - email@example.com; Rajini Ambalavanar - RAmbalavanar@umaryland.edu; Dong Wei - DWei@umaryland.edu;
Dean Dessem - firstname.lastname@example.org
* Corresponding author
Active regulation of gene expression in the nervous system plays an important role in the
development and/or maintenance of inflammatory pain. MicroRNA (miRNA) negatively regulates
gene expression via posttranscriptional or transcriptional inhibition of specific genes. To explore
the possible involvement of miRNA in gene regulation during inflammatory pain, we injected
complete Freund's adjuvant (CFA) unilaterally into the rat masseter muscle and quantified changes
in neuron-specific mature miRNAs in the trigeminal ganglion (TG). Real-time reverse-transcription
polymerase chain reaction revealed significant, but differential, downregulation of mature miR-10a,
-29a, -98, -99a, -124a, -134, and -183 in the ipsilateral mandibular division (V3) of the TG within 4
hr after CFA. In contrast, levels of tested miRNAs did not change significantly in the contralateral
V3 or the ipsilateral ophthalmic and maxillary divisions of the TG from inflamed rats, nor in the
ipsilateral V3 of saline-injected animals. The downregulated miRNAs recovered differentially to a
level equal to or higher than that in naive animals. Full recovery time varied with miRNA species
but was at least 4 days. Expression and downregulation of some miRNAs were further confirmed
by in situ hybridization of TG neurons that innervate the inflamed muscle. Although neurons of all
sizes expressed these miRNAs, their signals varied between neurons. Our results indicate that
miRNA species specific to neurons are quickly regulated following inflammatory muscle pain.
Inflammation associated with some pathologies may
develop allodynia or hyperalgesia defined as an over-reac-
tion to non-noxious or noxious stimuli, respectively [1,2].
Gene expression is an important molecular mechanism
underlying inflammatory pain since the measured steady-
state levels of mRNA and/or protein in pain/nociceptive
pathway in animal models are actively altered during the
development and maintenance of pain [2-6]. Our under-
standing of how individual genes are selectively regulated
during inflammatory pain is limited mostly to the regula-
tion of transcriptional control . MicroRNA (miRNA)
represents a group of small noncoding RNAs in 18~23
nucleotide sequences. These evolutionarily conserved
molecules mainly interfere with gene expression at post-
transcriptional levels and moderately promote RNA deg-
radation by acting on specific sequences in the 3'
untranslated region of target mRNA, while some of them
inhibit gene transcription by participating in chromatin
remodeling [7-9]. While many miRNAs have been
detected in the nervous system [10-12], their functional
significance has been restricted mostly to events involving
nervous system development [10,13-18]. Although miR-
Published: 8 June 2007
Molecular Pain 2007, 3:15doi:10.1186/1744-8069-3-15
Received: 4 May 2007
Accepted: 8 June 2007
This article is available from: http://www.molecularpain.com/content/3/1/15
© 2007 Bai et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Pain 2007, 3:15http://www.molecularpain.com/content/3/1/15
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NAs are present in mature neurons, their functionality
and regulation remain largely unexplored.
To explore the mechanism(s) underlying the gene altera-
tion during inflammatory pain and to investigate the
function of miRNA in the adult nervous system, we quan-
tified several neuronal miRNAs in a model of inflamma-
tory muscle pain. We injected CFA (150 µl, oil:saline =
1:1, Sigma, St. Louis, MO) unilaterally into the masseter
muscle of male rats (Sprague-Dawley, ~250 gr, Harlan,
Indianapolis, IN). This injection produced significant
mechanical allodynia while intramuscular injection of
saline did not [4,6]. After the development of allodynia,
we dissected the ophthalmic and maxillary divisions (V1/
2) and V3 from individual TGs. Tissues were combined
from two animals and cellular RNA was extracted for
miRNA quantification . This design is based on the
hypothesis that sensory neurons are a critical component
in pain/nociception pathway  and sensory neurons
innervating mandibular muscle have their perikarya
located in V3 . To quantify miRNA, we employed a
newly developed TaqMan real-time reverse-transcription
polymerase chain reaction (RT-PCR) assay (ABI, Foster
City, CA). This technology allows us to specifically meas-
ure selective mature miRNAs from nanogram amounts of
cellular RNA, thus making it possible to study small tis-
sues such as dissected TG . In the present study we
used the following criteria to limit miRNA number from
more than 400 identified molecules : First, miRNAs
expressed in TG were included. Seven miRNAs were
reported previously from TG . Second, those involved
in neuronal plasticity , one of cellular mechanisms
underlying inflammatory pain , were chosen. Third,
one member per miRNA family was examined [11,12,23].
Fourth, the amount of extracted RNA and the availability
of relevant TaqMan miRNA assays limited the number of
miRNA tested. Last, they are conserved among human
and rodents. In preliminary studies, we examined ten
miRNAs from a pool of RNA extracted from TG V3 (n =
16). We were able to detect miR-10a, -29a, -98, -99a, -
124a, -134, and -183, but not miR-122, miR-143, and
miR-153 even after 50 cycles of PCR, although they were
previously reported from TG via Northern analysis .
In parallel experiments, all assays produced robust signal
from brain RNA (data not shown).
We then analyzed the level of detectable miRNA species in
V3 of animals inflamed by CFA at various post-injection
time points. To correct sample loading and RT efficiency,
we normalized miRNA signal with the U6 RNA and the
These internal controls in our validation assays remained
stable along the tested time period (data not shown). Both
yielded similar results. But, since U6 RNA was detected by
the same type of TaqMan assay as miRNAs, in Fig. 1 we
show the results normalized by this small RNA. All tested
miRNAs were significantly downregulated within 4 hr
after CFA. The extent of downregulation can be catego-
rized into three groups: one retaining less than 5% of the
basal miRNA level in naïve animal (miR-10a, -98); one
maintaining 5~15% (miR-99, -124a, -183) and one show-
ing more than 25% left (miR-29a, -134). In the time
course of downregulation, miR-10a, -98, -99, and -124a
showed a long duration (~24 hr) of downregulation,
while miR-134 nearly fully recovered by this time (P >
0.05 compared to the basal level). By day 12, all tested
miRNAs were completely reversed to a level similar to or
higher than the basal level. We noticed that miR-29a, -99,
-124a, and -134 in inflamed animals reached a much
higher level than that in naïve animals. This phenomenon
was often seen during mRNA regulation  and the
rebounded change will be brought back to the base level
eventually, e.g. miR-29a and -134 in this study. In con-
trast, saline-injected animals did not show a significant
change in tested miRNAs in the ipsilateral V3 division.
The downregulation was also not seen in the contralateral
V3 or the ipsilateral V1/V2 divisions of the TGs in the
same CFA-injected animals (data not shown). More
importantly, from the same batch of RNA samples, calci-
tonin gene-related peptide mRNA was shown to be upreg-
Quantitative downregulation of mature miRNAs in the TG V3 following unilateral CFA injection
Quantitative downregulation of mature miRNAs in
the TG V3 following unilateral CFA injection. Total
RNA was extracted by the Absolutely RNA kit (Stratagene,
Lo Jolla, CA) and 10 ng of RNA was used to generate cDNA
using a TaqMan miRNA RT kit and TaqMan miRNA primers
specific to mature miRNA (ABI). Each TaqMan PCR reaction
contained cDNA derived from 0.88 ng RNA. Changes in
miRNA level were calculated by a ∆∆Ct method  and are
presented as percentage of control (the basal level in naïve
animal) in mean + s.e. from 6 to 12 samples. Each sample was
measured in triplicate. Sample differences were examined by
one-way ANOVA separately for each miRNA. *: P < 0.05
when compared to the control.
Molecular Pain 2007, 3:15http://www.molecularpain.com/content/3/1/15
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ulated 30 min after CFA, which correlates with the
development of mechanical allodynia . Therefore, we
believe that miRNA downregulation is specifically associ-
ated with the CFA-induced inflammation and allodynia.
Next, we addressed whether the neurons innervating the
injected muscle express these miRNAs and, if so, whether
these miRNAs respond to inflammation. We injected a
retrograde neuronal tracer, rhodamine-conjugated dex-
tran (Invitrogen, Carlsbad, CA), bilaterally into the mas-
seter muscle 5 days before the CFA injection . Four hrs
after the CFA injection, we perfused the animals (n = 6)
and examined miRNA in TGs using biotin-labeled locked
nucleic acid (LNA) probes (Exiqon, Vedbaek, Denmark)
in in situ hybridization. Neurons innervating the injected
muscles exhibited rhodamine signal and probes hybrid-
ized to miRNA were visualized by Cy3-conjugated strepta-
vidin (Fig. 2). Consistent with the real-time RT-PCR
results, downregulation of tested miRNAs was found in
TG neurons including rhodamine-positive cells that
innervate the inflamed muscle. In addition, miRNA sig-
nals were associated with neurons of all sizes although
large neurons seem to exhibit more signals. Interestingly,
LNA probes for miR-143 and -153 again did not produce
any positive signal (data not shown). These results
together with the TaqMan assays suggest that these
miRNA species are present at a very low level or not
expressed in TG V3 neurons.
To support the above observations, we tailed miR-134
and -143 LNA probes with digoxigenin-dUTP and viewed
miRNA signal in in situ hybridization with a colorimetric
method, which in general produces better cellular mor-
phology. As shown in Fig. 3, these experiments show a
downregulation of miRNA comparable to that obtained
by the fluorescent method. The stability of precipitated
color further confirmed the quantitative change in TG
neurons. Glial cells and other nonneuronal cells in TG did
not show detectable miR-134. Again, the miR-143 probe
did not reveal any signal in these experiments (data not
The LNA probe is virtually antisense to the mature miRNA
sequence that is present in both pre- and mature miRNA
in the cytosol . Therefore, the signal obtained from in
situ hybridization represents both types of molecules of a
specific miRNA. The results of the TaqMan assay and in
situ hybridization suggest that the downregulation
occurred either at both pre- and mature miRNA levels or
only at the mature miRNA level if the latter is the major
form in the TG.
The present study for the first time demonstrates miRNA
expression in the peripheral nervous system at the mature
miRNA level and with single cell resolution. Most impor-
miR-134 level in the ispi- and contralateral TG
miR-134 level in the ispi- and contralateral TG. The
LNA probe of miR-134 was tailed by digoxigenin-dUTP, and
visualized by a detection kit (Roche). Ipsi = ipsilateral; Contl
= contralateral. Red arrowhead: large neuron; blue arrow-
head: small neuron.
Distribution and downregulation of miRNA in TG
Distribution and downregulation of miRNA in TG.
TG tissues were obtained from animals inflamed by CFA for
4 hr and in situ hybridization was performed with 5' biotin
labeled LNA probes according to the protocol recom-
mended by the manufacturer (Exiqon). Bound probes were
detected by Cy3-streptavidin for green fluorescence while
the tracer rhodamine-conjugated dextran produced red fluo-
rescence. White arrowhead indicates tracer labeled cells.
Molecular Pain 2007, 3:15 http://www.molecularpain.com/content/3/1/15
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tantly, we observed that several miRNA molecules, likely
in the mature form, are regulated by an inflammatory irri-
tant and their changes are correlated with the develop-
ment of allodynia. Although the detailed mechanism
underlying this regulation remains unknown at this stage,
the RNA polymerase II (Pol II) is found to govern the tran-
scription of the most miRNA genes , and inflammation
is known to induce rapid expression or modification of
several transcription factors such as c-fos and CREB in
neurons [2,3,6]. These factors may negatively regulate Pol
II activity in neurons under certain conditions.
Discovery of miRNA downregulation provides a novel
view of the mechanism(s) underlying inflammatory pain.
Downregulation of miRNA releases the translation inhibi-
tion of target mRNAs, thus yielding more proteins that
may be relevant to the development and/or maintenance
of inflammatory pain. However, these initial studies only
demonstrated downregulation of a few selected miRNAs
in TG sensory neurons during the time when allodynia
occurred . Whether this miRNA downregulation is
mechanistically involved in inflammatory pain cannot be
addressed by the present study. How miRNA participates
in inflammatory pain relies, at least in part, on the eluci-
dation of their target mRNAs and/or on the impact of
manipulated levels of specific miRNA on nociception. The
former is a complex question. Even though several pro-
grams have been developed to predict the potential targets
for a given miRNA [23,25-27], systematic studies are
needed to thoroughly address this question.
The author(s) declare that they have no competing inter-
GB is responsible for initiation, experimental design, per-
formance of real-time RT-PCR assays, data analysis, and
drafting and finalizing the manuscript of this project. RA
contributed to experimental design, animal experiments
and RNA extraction as well as manuscript editing. DW
conducted in situ hybridization experiments and data
analysis. DD contributed to support, statistical analysis,
and manuscript editing. All authors have read and agreed
with the final manuscript.
This work is supported by NIH grants NS38077, DE15386 and DE016795.
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