Villa et al. Molecular Pain 2010, 6:89
http://www.molecularpain.com/content/6/1/89
MOLECULAR PAIN
RESEARCH
Open Access
Temporomandibular joint inflammation activates
glial and immune cells in both the trigeminal
ganglia and in the spinal trigeminal nucleus
Giovanni Villa1, Stefania Ceruti1, Matteo Zanardelli1, Giulia Magni1, Luc Jasmin2,3, Peter T Ohara3,
Maria P Abbracchio1*
Abstract
Background: Glial cells have been shown to directly participate to the genesis and maintenance of chronic pain in
both the sensory ganglia and the central nervous system (CNS). Indeed, glial cell activation has been reported in
both the dorsal root ganglia and the spinal cord following injury or inflammation of the sciatic nerve, but no data
are currently available in animal models of trigeminal sensitization. Therefore, in the present study, we evaluated
glial cell activation in the trigeminal-spinal system following injection of the Complete Freund’s Adjuvant (CFA) into
the temporomandibular joint, which generates inflammatory pain and trigeminal hypersensitivity.
Results: CFA-injected animals showed ipsilateral mechanical allodynia and temporomandibular joint edema,
accompanied in the trigeminal ganglion by a strong increase in the number of GFAP-positive satellite glial cells
encircling neurons and by the activation of resident macrophages. Seventy-two hours after CFA injection, activated
microglial cells were observed in the ipsilateral trigeminal subnucleus caudalis and in the cervical dorsal horn, with
a significant up-regulation of Iba1 immunoreactivity, but no signs of reactive astrogliosis were detected in the
same areas. Since the purinergic system has been implicated in the activation of microglial cells during
neuropathic pain, we have also evaluated the expression of the microglial-specific P2Y12 receptor subtype. No
upregulation of this receptor was detected following induction of TMJ inflammation, suggesting that any possible
role of P2Y12 in this paradigm of inflammatory pain does not involve changes in receptor expression.
Conclusions: Our data indicate that specific glial cell populations become activated in both the trigeminal ganglia
and the CNS following induction of temporomandibular joint inflammation, and suggest that they might represent
innovative targets for controlling pain during trigeminal nerve sensitization.
Background
Chronic pain is a pathological condition mainly associated with damage or dysfunction of peripheral and
central sensory pathways (neuropathic pain), or to tissue
inflammation (inflammatory pain) [1]. Despite efforts in
the last decades towards the understanding of its pathophysiology and the development of new drugs, chronic
pain still remains a difficult to manage and disabling
condition. The reason for this failure may be in part due
to the fact that most of the available drugs target neurons [2,3], whereas increasing evidence now indicates
* Correspondence: mariapia.abbracchio@unimi.it
1
Department of Pharmacological Sciences, Università degli Studi di Milano,
via Balzaretti 9, 20133 Milan, Italy
Full list of author information is available at the end of the article
that glial cells also play an important role in the generation and maintenance of chronic pain [4-7]. During
pathological pain states, in the central nervous system
(CNS) both microglial cells and astrocytes become activated and start releasing proinflammatory signals which
are responsible for the hyperexcitability of nociceptive
pathways, thereby leading to the development of hyperalgesia and allodynia [8-11]. Accordingly, the pharmacological inhibition of glial cell function effectively
attenuates the development of both neuropathic [12,13]
and inflammatory pain [14].
Accumulating evidence suggests that glial cells of sensory ganglia also participate in the development and
maintenance of chronic pain conditions [15,16]. Following stimulation of neurons in the dorsal root ganglia
© 2010 Villa 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.
Villa et al. Molecular Pain 2010, 6:89
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(DRG), satellite glial cells (SGCs) wrapped around neuronal bodies [17] become activated and increase neuronal excitability by releasing inflammatory mediators
such as tumour necrosis factor-a (TNFa) [18]. Following sciatic nerve injury, an increased expression of interleukin (IL)-6 [19] and of other pro-inflammatory
cytokines [20] is also detected in SGCs. Moreover, DRG
neurons release chemokines that trigger macrophage
invasion, suggesting that the latter cell population is also
involved in the regulation of chronic pain [21-23].
The trigeminal ganglion (TG) is the location of primary afferent neurons for sensing and relaying nociceptive sensations associated with painful conditions such
as dental pain, trigeminal neuralgia, and temporomandibular disorders [24]. Cutaneous inflammation triggers
the activation of SGCs in the TG, leading to the release
of the pro-inflammatory cytokine IL-1b [25], which in
turn increases the firing activity of nociceptors [26].
Interestingly, the selective silencing of the SGCs proteins
connexin 43 or glutamine synthase in the TG significantly reduced the hyperalgesia associated with the
chronic constriction of the infra-orbital nerve [27], thus
suggesting that SGCs are key determinants of chronic
pain intensity. A well-established model of inflammatory
pain, which shares several characteristics with migraineassociated TG sensitization [28,29], is based on the
injection of pro-inflammatory mediators (e.g., capsaicin
or Complete Freund’s Adjuvant, CFA) in the temporomandibular joint (TMJ). It was shown that TMJ inflammation potentiates the excitability of nociceptive
neurons in both the TG [26] and in the spinal trigeminal nucleus [30], and leads to increased neuron-to-SGC
communication within the TG [31,32], but to date, no
studies have explored the reaction of glial cells in the
whole trigeminal-spinal system following the induction
of TMJ inflammation. Therefore, in the present study
we have characterized the reaction of peripheral nervous
system (PNS) and CNS glial cells to the injection of
CFA into the rat TMJ.
Results
Development of mechanical allodynia and inflammation
after injection of CFA into the TMJ
First of all, to evaluate inflammation following CFA
injection, we measured the TMJ extravasation of the
Evans’ blue dye injected into the tail vein. While there
was no difference in the dye concentration in the TMJs
of rats injected with saline (0.07 ± 0.07 μg/ml for the
ipsilateral side vs. 0.07 ± 0.06 μg/ml for the contralateral
side, p = 0.977; n = 7 animals) (Figure 1A), a significantly greater amount of dye was extracted from the
ipsilateral TMJ of CFA-injected rats, both at 24 h p.i.
(0.76 ± 0.17 μg/ml vs. 0.13 ± 0.05 μg/ml for ipsi- and
contralateral tissue, respectively, p < 0.01; n = 6
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Figure 1 Injection of CFA into the TMJ produces plasma
extravasation and mechanical allodynia. (A) The development of
inflammation was analyzed by injecting the Evans’ blue dye through
the tail vein. A significantly higher amount of dye was extracted
from the ipsilateral TMJs of CFA-injected rats compared to the
contralateral side at both 24 h and 72 h p.i. Since no significant
differences between saline-injected rats at 24 h and 72 h have been
observed, data have been pooled together, and shown here as
“saline”. (B) Rats were tested for the development of mechanical
allodynia by probing the contralateral (contra) and ipsilateral (ipsi)
orofacial regions with von Frey filaments before (pre) and post
injection of saline or CFA into the TMJ. The head withdrawal
threshold force, in grams (g), was measured. Y-axis = log10 scale. **
p < 0.01, and * p < 0.05 compared to the contralateral side of CFAinjected rats, # p < 0.01 compared to the contralateral tissue; oneway ANOVA.
animals), and, to a lesser extent, at 72 h p.i. (0.43 ± 0.07
μg/ml vs. 0.07 ± 0.03 μg/ml for ipsi- and contralateral
tissue, respectively, p < 0.01; n = 7 animals) (Figure 1A).
The development of allodynia, due to trigeminal sensitization secondary to the primary inflammatory event,
was then evaluated by probing the orofacial regions of
saline- or CFA-injected animals with von Frey filaments.
Baseline values for mechanical threshold were determined in non-inflamed rats. Mean head withdrawal
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thresholds were measured from the left and right orofacial regions (16.11 ± 2.06 g for the right side and 15.50 ±
1.73 g for the left side; n = 12 animals) (Figure 1B). Rats
were then injected with either saline or CFA into the left
TMJ and tested for their pain behavior. A significantly
lower threshold to innocuous mechanical stimuli was
measured for the ipsilateral side of CFA injected rats,
starting 24 h post injection (p.i.) (Figure 1B and Table 1).
No significant changes between the ipsi- and the contralateral mechanical thresholds were observed in control
saline-injected animals (Figure 1B and Table 1).
These results confirm that the injection of CFA into
the TMJ induces a persistent local inflammation, associated with the development of mechanical allodynia.
SGCs and macrophages are selectively activated in
trigeminal ganglia following TMJ inflammation
We next evaluated the morphological and biochemical
consequences of the induction of TMJ inflammation in
the TG, with particular focus on SGCs [33]. Previous studies have reported that either tooth pulp injury or TG
inflammation induces SGCs hypertrophy, with increased
expression of GFAP [25,34,35], which is considered to be
a marker of reactivity and activation for this particular
type of glia. In our experimental model, very low levels of
GFAP immunoreactivity were observed in the contralateral side of CFA injected rats, as measured by counting
the number of TG neurons encircled by GFAP-positive
(GFAP+) SGCs (Figure 2A, B). As expected [25], a large
increase in the number of GFAP-encircled neurons was
observed in the ipsilateral TG at both 24 h (Figure 2A’)
and 72 h p.i. (Figure 2B’). From 24 h p.i. the number of
GFAP-encircled neurons was higher in the ipsilateral V3mandibular (29.38 ± 2.20 in the ipsilateral side vs. 5.99 ±
0.94 in the contralateral side, p < 0.001; n = 7 animals),
and V2-maxillary (28.79 ± 2.30 vs. 4.92 ± 1.00, p < 0.001;
n = 7 animals) division of the trigeminal nerve. The same
effect was also observed in the V1-ophthalmic division,
although to a lesser extent (12.83 ± 1.24 vs. 3.88 ± 0.69,
p < 0.001; n = 7 animals) (Figure 2C-E). At 72 h p.i. a
reduction in GFAP staining was detected compared to
the values at 24 h, but it was still significantly higher with
respect to the contralateral side or controls (p < 0.05 and
p < 0.001 for the V1 and V2-3 division respectively; n = 7
animals) (Figure 2C-E). No differences in the number of
GFAP-encircled neurons were found between the ipsiand contralateral TGs in saline-injected rats (p = 0.805,
p = 0.319, p = 0.432 for the V1, V2, V3 division respectively; n = 7 animals) (Figure 2C-E).
Macrophages have been reported to infiltrate the DRG
following sciatic nerve damage or hindpaw inflammation
[36,37]. We therefore evaluated whether a similar effect
also takes place in the TG after the development of
TMJ inflammation. Surprisingly, in any of the three divisions of the ipsilateral TG no changes in the number of
Iba1+ resident macrophages and no difference in their
mean cell size were observed at either 24 h or 72 h following CFA injection (see Figure 3, panels A-B’ for
representative images of Iba1 staining, and panels C-E
and F-H for quantification of Iba1 cell number and of
their average cell size, respectively). Nevertheless, clear
signs of macrophagic activation were detected by using
an antibody directed against the lysosomal antigen ED1,
a marker of activated macrophages (Figure 4A-B’)
[38-40]. Quantification of results has been performed by
both a densitometric analysis of ED1 immunostaining
(Figure 4C-E), and by counting the number of ED1 +
cells in identical areas in each coverslip (Figure 4F-H).
Both analyses demonstrated a significant increase of
ED1 immunostaining in the three divisions of ipsilateral
TG with respect to the contralateral side at 24 h after
CFA injection, with the highest effect detected in the
V3-mandibular division (densitometric analysis: 111.39
± 16.71 pixels for the ipsilateral side vs. 36.44 ± 10.73
pixels for the contralateral side, p < 0.01, n = 6 animals;
number of ED1+ cells: 18.19 ± 2.10 cells for the ipsilateral side vs. 8.18 ± 0.96 cells for the contralateral side,
p < 0.01; n = 7 animals; Figure 4E, H). These effects
Table 1 Mean head withdrawal threshold values after saline or Complete Freund’s Adjuvant (CFA) injection in the
temporomandibular joint.
Stimulus
Time post injection
Ipsilateral (g)
Contralateral (g)
n° of rats
p value (ipsi vs. contra)
p value (ipsi vs. baseline)
baseline
-
15.5 ± 1.73
16.1 ± 2.06
12
-
-
saline
4h
15.0 ± 0.00
20.5 ± 5.50
4
0.423
0.813
24 h
12.7 ± 2.33
17.0 ± 4.73
4
0.457
0.395
48 h
17.0 ± 4.73
17.0 ± 4.73
4
1.000
0.845
72 h
16.3 ± 5.24
17.0 ± 4.73
4
0.929
0.962
4h
10.1 ± 2.59
15.1 ± 3.07
8
0.231
0.088
24 h
48 h
6.94 ± 1.45
5.33 ± 0.67
14.0 ± 2.47
10.5 ± 0.96
8
8
< 0.05
< 0.01
< 0.01
< 0.01
72 h
5.67 ± 1.20
12.7 ± 1.48
8
< 0.01
< 0.01
CFA
g: grams; ipsi: ipsilateral side; contra: contralateral side.
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Figure 2 GFAP immunoreactivity is increased in SGCs following induction of TMJ inflammation. (A, B) Few GFAP+ SGCs (red) encircling
NeuN+ neurons (green) were detected in the contralateral (CONTRA) TG of CFA-injected rats, or in TGs from saline-injected rats (see below). A
significant increase in the number of GFAP-encircled neurons was instead observed in the ipsilateral (IPSI) TG starting at 24 h post CFA injection
(A’, B’). Nuclei were labeled with the Hoechst 33258 dye (blue). Scale bars: 20 μm. (C-E) The number of GFAP-encircled neurons was increased in
all 3 divisions (V1-ophthalmic, V2-maxillary, V3-mandibular) of ipsilateral (ipsi) TG. Similar, although smaller, changes were observed at 72 h p.i.
Since no significant differences between saline-injected rats at 24 h and 72 h have been observed, data have been pooled together, and shown
here as “saline”. Data are expressed as number of GFAP-encircled neurons per counting field at 40x magnification, and refer to 2 independent
experiments. *** p < 0.001 and * p < 0.05 compared to the contralateral side; one-way ANOVA.
persisted at 72 h p.i., although to a lesser extent
(Figure 4). These results suggest that, early after induction of TMJ inflammation, there is no recruitment of
new inflammatory cells from the bloodstream, but
rather a strong activation of local resident macrophages.
Microglial cells, but not astrocytes, are activated in the
spinal trigeminal nucleus following TMJ inflammation
Although previous studies reported that CNS glial cells
(astrocyte and microglial cells) become activated following CFA-induced sciatic nerve inflammation [41-43], no
studies on the role of these cells after inflammatory sensitization of the TMJ are available.
The primary afferent fibers of the trigeminal nerve terminate in the spinal trigeminal nucleus of the brainstem,
which extends through the pons and medulla, and
finally overlaps with the dorsal horn of the cervical
spinal cord [44]. Moving along its rostro-caudal axis, the
spinal trigeminal nucleus can be subdivided in subnucleus oralis, subnucleus interpolaris, and subnucleus
caudalis [44].
Immunoreactivity levels for Iba1 (a marker for microglial cells) and GFAP (a marker for astrocytes) in the
different regions of the spinal trigeminal nucleus were
thus evaluated in saline and CFA-injected rats. As
shown in Figure 5, in saline-injected rats no changes in
Iba1 immunoreactivity were observed between the
contra- and ipsi-lateral sides of either the trigeminal subnucleus caudalis of the medulla oblongata (p = 0.836; n =
8 animals) (Figure 5A) or of the dorsal horn of the cervical spinal cord (p = 0.918; n = 8 animals) (Figure 5C).
Seventy-two hours after CFA administration, Iba1 immunoreactivity was significantly up-regulated in the dorsal
laminae of the trigeminal subnucleus caudalis (normalized values: 1.51 ± 0.13 pixels for the ipsilateral side vs.
0.90 ± 0.06 pixels for the contralateral side, p < 0.01; n =
8 animals) (Figure 5A). Moreover, in the ipsilateral side
microglial cells displayed shorter and thicker ramifications, a typical characteristic of activated microglia, when
compared to the fine processes of microglia in the contralateral side (Figure 5B’, B’’). Similar changes were also
observed in the dorsal horn of the cervical spinal cord
(normalized values: 1.80 ± 0.21 pixels for the ipsilateral
side vs. 1.00 ± 0.12 pixels for the contralateral side, p <
0.01; n = 7 animals) (Figure 5C, D).
Conversely, no reactive astrogliosis was detected in the
spinal trigeminal nucleus, both in terms of GFAP immunoreactivity (p = 0.933 and p = 0.779 for the trigeminal
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Figure 3 No changes in the number of resident TG macrophages are induced by injection of CFA in the TMJ. Twenty-four (A, A’) or 72
hours (B, B’) after CFA injection, Iba1 immunostaining (green) of contralateral (CONTRA) and ipsilateral (IPSI) TG showed no morphological
changes of Iba1+ macrophages. Nuclei were labeled with the Hoechst 33258 dye (blue). Scale bars: 20 μm. No differences in either the number
(C-E) or in the average cell size (F-H) of Iba1+ macrophages were detected in any of the three TG divisions in inflamed compared to salineinjected rats. Since no significant differences between saline-injected rats at 24 h and 72 h have been observed, data have been pooled
together, and shown here as “saline”.
subnucleus caudalis at 24 h and 72 h respectively; p =
0.156 and p = 0.364 for the cervical dorsal horn at 24 h
and 72 h respectively; n = 7 animals) (Figure 6A, C),
and of the morphology of GFAP+ astrocytes (Figure 6B,
D). We conclude that, during the sub-acute phase of
CFA-induced TMJ inflammation, microglial cells, but
not astrocytes, are selectively activated in the CNS
regions relaying nociceptive information from the TG.
The purinergic P2Y12 receptor is selectively expressed by
microglial cells in the CNS, but it is not up-regulated
following TMJ inflammation
ATP and other extracellular nucleotides participate in pain
transmission under both normal and pathological
conditions [45-47]. Their receptors (namely the ligandgated P2X receptors, P2X1-7, and the metabotropic G protein-coupled P2Y receptors, P2Y 1,2,4,6,11,12,13,14 ) are
expressed not only by sensory neurons [48,49], but also by
all the different glial cell populations involved in nociception, including SGCs [18,50], macrophages [51], astrocytes
[52], and microglia [53]. In particular, in this latter cell
population the P2Y12 receptor subtype was shown to be
up-regulated following nerve injury, and its pharmacological or biotechnological inhibition prevented the development of mechanical allodynia [54,55]. On this basis, we
analyzed the possible changes of P2Y12 receptor expression in our inflammatory model. Using a specific antibody
directed against the C-terminal domain of the rodent
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Figure 4 Injection of CFA in the TMJ activates resident TG macrophages. (A-B’) ED1 immunostaining (green) of activated macrophages in
the V3-mandibular TG division, showing upregulation at both 24 h (A, A’) and 72 h (B, B’) post CFA injection. Nuclei were labeled with the
Hoechst 33258 dye (blue). Scale bars: 20 μm. (C-E) Densitometric analysis of ED1 immunoreactivity, showing that, at 24 h post CFA injection, a
significant upregulation of ED1 staining was detected in any of the three TG divisions, and that a lower, although not statistically significant,
effect was also present at 72 h. (F-H) Results from densitometric analysis were fully confirmed by counting the number of ED1+ cells in an
identical area for each coverslip. Since no significant differences between saline-injected rats at 24 h and 72 h have been observed, data have
been pooled together, and shown here as “saline”. Data are expressed as number of ED1+ macrophages per counting field at 40× magnification,
and refer to 3 independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the contralateral side; one-way ANOVA.
P2Y12 receptor [56], we were not able to detect any staining within the TG of control animals (Figure 7A). This
finding contrasts with previous reports indicating the presence of P2Y12 receptor mRNAs in both rat DRGs [57]
and mouse TG [50]. Interestingly, in the same tissue sections P2Y12 receptor immunoreactivity was detected at the
boundary between the trigeminal nerve root (i.e., the PNS)
and the CNS (Figure 7B), thus indicating that the expression of this receptor subtype is probably restricted to cells
of the CNS. Furthermore, while P2Y12 receptor and Iba1
immunostaining was colocalized in CNS microglial cells
(Figure 7B, yellow arrows), Iba1+ macrophages resident in
the trigeminal nerve root were P2Y12-negative (Figure 7A
and Figure 7B, red arrows). In the brainstem, no P2Y12
receptor expression was observed in either GFAP+ astrocytes (Figure 7C), or NeuN+ neurons (Figure 7D). Indeed,
the P2Y12 receptor subtype was only seen in Iba1+ microglial cells (Figure 7E, E’), confirming previous reports indicating this receptor expressed by CNS microglia [54-56].
We next evaluated whether P2Y12 receptor levels in
the spinal trigeminal nucleus were affected by CFA
injection into the TMJ or not. Despite the observed
upregulation of Iba1 immunoreactivity and the
morphological changes observed in microglial cells (see
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Figure 5 TMJ inflammation induces microglial activation in the spinal trigeminal nucleus. (A, C) Densitometric analysis of Iba1
immunoreactivity (number of immuno-positive pixels, see Methods) showing a significant increase in Iba1 immunoreactivity in the ipsilateral side
of both the trigeminal subnucleus caudalis in the medulla oblongata (delimited by the dotted line; A) and the dorsal horn of the cervical spinal
cord (C) 72 h after CFA injection. The mean values of pixel intensity have been normalized to the values obtained from the contralateral side of
saline injected rats, set to 1.0. Since no significant differences between saline-injected rats at 24 h and 72 h have been observed, data have been
pooled together, and shown here as “saline”. ** p < 0.01 compared to the contralateral side; one-way ANOVA. (B-B’’, D-D’’) Resting microglial cells (i.
e., ramified cells with fine processes) were detected in the contralateral side of the trigeminal subnucleus caudalis (B’) and of the cervical dorsal
horn (D’), whereas activated microglial cells (i.e. cells with thicker ramifications) were observed ipsilaterally (B’’, D’’). Scale bars: 20 μm.
above), no increase in P2Y12 receptor immunoreactivity
in the ipsilateral side of CFA-injected rats was
observed in either the trigeminal subnucleus caudalis
(p = 0.957 and p = 0.968 at 24 h and 72 h respectively;
n = 7 animals) (Figure 8A-B’’) or in the cervical dorsal
horn (p = 0.734 and p = 0.923 at 24 h and 72 h
respectively; n = 7 animals) (Figure 8C-D’’). Taken
together, these results demonstrate that the expression
of the purinergic P2Y 12 receptor, which has been
implicated in some forms of pain sensations, is not
modified in the subacute reaction of CNS glial cells to
TMJ inflammation.
Discussion
In both the CNS and PNS, glial cells have been shown
to actively participate in the genesis and maintenance of
chronic pain conditions, and might therefore represent
innovative targets for the development of new therapeutic approaches to pain management [4,7,9,11,27]. Most
of the studies investigating the role of glial cells in
inflammatory pain have been performed in animal models of sciatic nerve sensitization, such as the induction
of hindpaw inflammation [14,42,58,59] or of joint monoarthritis [41,43], but only limited information on the
role of trigeminal SGCs during TMJ inflammation is
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Figure 6 TMJ inflammation does not affect the activation state of GFAP+ astroglial cells in the CNS. (A, C) Densitometric quantification of
GFAP immunoreactivity in the trigeminal subnucleus caudalis (A) and the cervical dorsal horn (C) revealed no changes between the contralateral
(contra) and the ipsilateral (ipsi) sides of CFA inflamed rats. The mean values of pixel intensity have been normalized to the contralateral side of
saline injected rats, set to 1.0. Since no significant differences between saline-injected rats at 24 h and 72 h have been observed, data have been
pooled together, and shown here as “saline”. (B-B’’, D-D’’) No changes in astroglial cell morphology in the trigeminal subnucleus caudalis
(delimited by the dotted line; B-B’’) and the cervical dorsal horn (D-D’’) were seen. Scale bars: 20 μm.
available [25,31]. Moreover, no characterization of glial
cells activation along the whole trigeminal-spinal system
is currently available. Thus, in the present study we
have examined the response of TG and CNS glial cells
following injection of the pro-inflammatory agent CFA
in the rat TMJ.
TG glial and immune cells response to TMJ inflammation
Here, we demonstrate that CFA injection into the TMJ
produces a significant increase in GFAP expression in
SGCs in the TG, thus confirming the reaction of glial cell
to the administration of a pro-algogenic stimulus and
validating GFAP as a useful marker of trigeminal sensitization [25,34,35]. The key role of SGCs in the development
and maintenance of chronic pain has been demonstrated
by their increased expression and release of IL-1b [25],
TNFa [18], as well as augmented gap junction-mediated
cell coupling [31,60] following nerve injury. All together,
these changes are associated with increased excitability of
both primary afferents and CNS neurons, leading to the
development of hyperalgesia and allodynia [16,33].
We also provide new evidence on the behavior of TG
macrophages under inflammatory conditions. In particular, following induction of TMJ inflammation, a strong
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footpad or of the knee joint [37,62] which the authors
attributed to macrophage infiltration. Although the total
number of immune cells was not evaluated in these studies, and therefore activation of resident cells cannot be
excluded, it may well be that the responses evoked in
DRG and TG by the same inflammatory event are different, suggesting high regional and time specificity.
CNS glial cells response to TMJ inflammation
Figure 7 P2Y12 receptor immunoreactivity is absent in the TG,
but selectively localized to microglial cells in the brainstem. (A)
In the TG, no staining for the P2Y12 receptor subtype (green) was
detected in Iba1-expressing macrophages (red). (B) At the trigeminal
nerve root (i.e., the PNS/CNS boundary) Iba1+/P2Y12+ microglial cells
were instead observed (yellow arrows). (C-E’) In the brainstem, no
colocalization between P2Y12 receptor (green) and either the
astrocytic marker GFAP (red; C), or the neuronal marker NeuN (red;
D) was observed. P2Y12 receptor immunoreactivity was instead
specifically found on Iba1+ microglial cells (red; E, E’). Nuclei were
labeled with the Hoechst 33258 dye (blue). Scale bars: 20 μm.
upregulation of ED1 (a specific marker for activated
macrophages) [38] in the ipsilateral TG was found.
Because there was no increase in either the number or
the average cell size of Iba1+ macrophages, and based
on the fact that Iba1 is expressed by both resting and
activated microglia/macrophages [61], we conclude that
CFA injection does not trigger macrophages infiltration
into the trigeminal perineuronal regions from the bloodstream, but rather modulates the activation state of resident immune cells. These cells are, in turn, involved in
the development of TG sensitization following TMJ
inflammation. This is at variance from previous reports
indicating an increase in ED1 immunoreactivity in the
DRG following CFA-induced inflammation of the
Microglial cells, the resident macrophagic population in
the CNS, have been crucially implicated in the initiation
and modulation of certain chronic pain states [63]. For
instance, in various neuropathic pain models, activated
microglia was shown to release proinflammatory cytokines and other substances that facilitate pain transmission [64,65]. Moreover, several drugs acting as glial cell
inhibitors (e.g., propentofylline, pentoxifylline and minocyclin) eventually suppress the development of neuropathic pain by decreasing both microglial activation and
cytokine release [12,13].
Here we report for the first time that injection of CFA
into the TMJ induces a significant ipsilateral activation
of microglial cells, both in the dorsal laminae of the trigeminal subnucleus caudalis in the medulla oblongata
and in the dorsal horn of the cervical spinal cord, which
are the regions receiving the mandibular fibers of the
trigeminal nerve [44], and represent key stations for the
integration of temporomandibular painful sensations.
Our data are in agreement with previous papers indicating an increased expression of microglial cell markers
in the lumbar spinal cord, associated with the appearance of cells having an activated morphology, in CFAinduced ankle or tibio-tarsal joint monoarthritis in rats
[41,43]. This confirms that the induction of deep articular inflammation represents a potent trigger for CNS
microglial activation. On the other hand, conflicting
results have been obtained following induction of subcutaneous hindpaw inflammation by CFA [14,42,58]. Overall, the current hypothesis is that other inflammatory
substances rather than CFA, like formalin or zymosan,
can activate spinal cord microglia following their hindpaw injection, suggesting a strict dependence of microglia recruitment from both the inflammatory stimulus
and the injection site.
We also examined astrocyte activation in the brainstem following TMJ inflammation. At variance from
previous reports on sciatic nerve inflammation [42,43],
we did not detect any evidence of astroglial activation.
This divergence could be due to the fact that the latter
studies showed astroglial activation occurred at later
time points (11 and 14 days) following the inflammatory insult, thus suggesting that astrocytes are not
involved in the first sub-acute phases of tissue
sensitization.
Villa et al. Molecular Pain 2010, 6:89
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Figure 8 P2Y12 receptor levels in the CNS are not affected by the induction of TMJ inflammation. (A, C) Densitometric quantification of
P2Y12 receptor immunoreactivity in both the trigeminal subnucleus caudalis (A) and the cervical dorsal horn (C) revealed no changes between
the contralateral (contra) and the ipsilateral (ipsi) sides of inflamed rats. The mean values of pixel intensity have been normalized to the
contralateral side of saline injected rats, set to 1.0. Since no significant differences between saline-injected rats at 24 h and 72 h have been
observed, data have been pooled together, and shown here as “saline”. (B, D) Immunostaining of the P2Y12 receptor subtype in the trigeminal
subnucleus caudalis (delimited by the dotted line; B-B’’) and the cervical dorsal horn (D-D’’). Scale bars: 20 μm.
No evidence for upregulation of the P2Y12 receptor
subtype in the sub-acute phases following TMJ
inflammation
The purinergic P2Y12 receptor is a Gi-coupled metabotropic receptor specifically activated by extracellular
ADP [52] that has been shown to control the chemotaxis of CNS microglial cells in response to nucleotides
[56,66]. Moreover, microglial P2Y12 receptors have been
demonstrated to increase at both the mRNA and protein level in the ipsilateral lumbar spinal cord following
nerve injury in rats [54,55], and both the pharmacological and biotechnological blockade of this receptor
prevented the development of neuropathic pain hypersensitivity [54,55]. Furthermore, P2Y12 knock-out mice
displayed deficiency in the development of tactile allodynia following nerve injury, despite normal basal mechanical sensitivity [55]. Therefore the P2Y12 receptor can be
considered as a key player in controlling microglia-associated development of neuropathic pain, thus representing a possible therapeutic target for treating chronic
pain disorders. For these reasons we checked for its possible modulation in our inflammatory pain model. By
utilizing a custom-made antibody whose specificity was
already successfully validated in P2Y12 -deficient mice
Villa et al. Molecular Pain 2010, 6:89
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[56], no P2Y12 immunostaining was detected on TG sections, although we have previously detected P2Y 12
mRNA in mouse TG [50]. Interestingly, staining for the
P2Y12 receptor was observed at the boundary between
the trigeminal nerve root and the CNS, and in Iba1+
cells of the brainstem. This observation correlates with
previous reports indicating that, unlike other extracellular nucleotide receptors, such as the P2X4 , P2X7 , and
P2Y6 receptor subtypes [53,67], P2Y12 receptor expression is restricted to CNS microglia [56,68]. Quite surprisingly, we detected no changes in microglial P2Y 12
receptor levels after the injection of CFA in the TMJ,
suggesting that this receptor is not up-regulated during
the first phases of tissue hypersensitivity following an
inflammatory stimulus. It is worth mentioning that in a
different model of inflammatory pain (i.e., the injection
of CFA in the hindpaw) the intraperitoneal administration of the P2Y 12 receptor antagonist 2,2-dimethylpropionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2(2,2-dimethyl-propionyloxymethyl)-propylester
(MRS2395) significantly alleviated the mechanical hypersensitivity [69], but authors did not evaluated the possible changes in P2Y 12 receptor expression in their
experimental model. Therefore, it may well be that,
despite the lack of detectable receptor up-regulation,
P2Y 12 receptor targeting might be beneficial also for
treating other chronic inflammatory pain states involving the sensitization of the trigeminal-spinal system, as
in our experimental model.
Conclusions
In the present study we have characterized glial cell
reaction in the nervous system in a rat model of TMJ
inflammation. Specifically, we have shown that CFA
injection is associated with activation of SGCs and
macrophages in the TG, as well as of microglial cells in
the spinal trigeminal nucleus, with no signs of reactive
astrogliosis. Therefore, our results clearly demonstrate a
correlation between the activation of both TG and CNS
glial cells and inflammatory pain. Additional studies are
now needed in order to demonstrate that the observed
glial activation in one or both sites plays a direct role in
either the initiation or the maintenance of the pain
behavior, and to suggest glial cells as possible innovative
pharmacological targets for treating trigeminalassociated pain.
Methods
Animals
Experiments were performed on adult male SpragueDawley rats (200 - 250 g; Charles River Lab, Calco,
Milan, Italy). Animals were housed under controlled
conditions (temperature 22 ± 2°C; relative humidity
50 ± 10%; artificial light 12 h light/dark cycle, lights on
Page 11 of 14
at 7 AM). All animals had access to both distilled water
and standard diet ad libitum. The study has been
approved by the Council of the Department of Pharmacological Sciences of the Università degli Studi di
Milano, Milan, Italy, and was carried out in accordance
with National and European regulations regarding the
protection of animals used for experimental and other
scientific purposes (D.M. 116192; 86/609/EEC), as well
as following the ethical guidelines of the International
Association for the Study of Pain (IASP) [70].
Induction of TMJ inflammation
The sub-chronic TMJ inflammation was induced by
injecting 50 μl of CFA (Sigma, Milan, Italy) oil/saline
(1:1) emulsion into the left TMJ capsule, under isoflurane anesthesia. Control rats were injected with saline
(0.9% NaCl). The TMJ capsule was identified by palpating the zygomatic arch and condyle, and the injection
was delivered by advancing a 27-gauge needle medioanteriorly through the skin immediately below to the posteroinferior border of the zygomatic arch until it
entered into the joint capsule [71]. Then, CFA or saline
was slowly injected over two minutes.
Behavioral test
Mechanical allodynia was measured by a previously
described protocol with some minor modifications [72].
Unrestrained rats were trained to stay in position and to
be probed with von Frey filaments (North Coast Medical, Morgan Hill, CA, USA) at least one week before the
injection of CFA. The left and right orofacial skin
regions, near to the center of the vibrissa pad, were
tested. An ascending series of von Frey filaments was
used. The starting filaments corresponded to log unit
4.31 (force: 2 grams) and 3.22 (force: 0.16 grams) for
control and inflamed animals, respectively. Each filament was tested five times with an interval of a few seconds. The response threshold was defined as the lowest
force required to elicit at least three head withdrawal
responses out of five tests. The elapsed time between
the applications of a new filament was 2 minutes. All
experiments were carried out in a quiet room between
the 8.30 AM and 1.00 PM, in order to avoid diurnal
variations.
Measurement of Evans’ blue dye extravasation
Evans’ blue dye (5 mg/kg, 0.3% solution) was injected
into the tail vein [73], 10 minutes before perfusion (see
below). Ipsi- and contra-lateral (with respect to the side
of CFA injection) TMJs were then dissected, cut into
small blocks and incubated overnight at room temperature (RT) in a 7:3 (vol/vol) mixture of acetone and
35.2 mM sodium sulfate on a shaking table. Samples
were then centrifuged, the supernatant separated, and
Villa et al. Molecular Pain 2010, 6:89
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dye absorbance determined in a spectrophotometer at
620 nm. Evans’ blue dye concentrations, in μg/ml, were
extrapolated from a best-fit line calculated from a standard curve, prepared from a series of supernatants
extracted from the TMJs of naïve animals, and mixed
with serial dilutions of the Evans’ blue dye.
Tissue processing
Twenty-four and 72 hours after CFA injection, rats were
anesthetized with intraperitoneal injection of 400 mg/kg
chloral hydrate and transcardially perfused with 4% formalin fixative. Both the intact brain and TGs were then
excised, postfixed in 4% formalin for 60-90 min, and
cryoprotected in 30% sucrose for at least 48 hours. The
left and right TG from each animal were embedded
together in mounting medium (OCT; Tissue Tek,
Sakura Finetek, Zoeterwoude, The Netherlands), and cut
longitudinally on a cryostat at 15 μm thickness. Brainstems were separated from the rest of the brain, and
marked ventrally on the contralateral side to subsequently identify tissue orientation. Transverse 40 μm
thick free-floating sections were then cut on a cryostat.
Immunohistochemistry
Free-floating brainstem sections, or on-slide TG sections, were incubated for 45 min at RT in PBS containing 10% normal goat serum (Sigma-Aldrich, Milan,
Italy) and 0.1% Triton X-100 (Sigma-Aldrich), and then
overnight at RT with the following primary antibodies:
rabbit anti-glial fibrillary acidic protein (GFAP, 1:600;
Dako, Milan, Italy), mouse anti-NeuN (1:500; Millipore,
Vimodrone, Italy), rabbit anti-ionized calcium binding
adaptor molecule 1 (Iba1, 1:800; Biocare Medical, Space
Import-Export, Milan, Italy), rabbit anti-P2Y12 receptor
polyclonal antiserum (1:1,500; a generous gift by Prof.
David Julius, University of California San Francisco, CA,
USA), and mouse anti-ED1 (1:200; Serotec, Space
Import-Export).
For fluorescence analysis, sections were then rinsed
three times with PBS, and incubated for 1 h at RT
with goat anti-rabbit and goat anti-mouse secondary
antibodies conjugated to AlexaFluor®488 or AlexaFluor®555 fluorochromes (1:600; Molecular Probes,
Invitrogen, Milan, Italy). Nuclei were subsequently
labeled with the fluorescent dye Hoechst 33258
(1:10,000 in PBS; Molecular Probes). Slides were finally
washed, mounted with Fluorescent Mounting Medium
(Dako), and examined with a laser scanning confocal
microscope (LSM 510; Zeiss, Jena, Germany). Images
were acquired and analyzed using the LSM Image
Browser software (Zeiss).
For light microscopy, sections were incubated for 1 h
at RT with an anti-rabbit biotinylated secondary antibody (1:500; PerkinElmer, Monza, Italy), and then with
Page 12 of 14
a horseradish peroxidase (HRP)-conjugated streptavidin
(1:400, 45 min at RT; PerkinElmer). To visualize the formation of the antibody-antigen complex, the nickel-3,3’diaminobenzidine (Sigma-Aldrich) protocol was used.
Sections were mounted with DPX (Sigma-Aldrich), and
analyzed with an inverted microscope (Axiovert 200;
Zeiss) equipped with a color CCD camera (AxioCam
HRc; Zeiss), connected to a PC computer equipped with
the software Axiovision (Zeiss).
Non-specific staining was evaluated on sections where
the primary antibodies were omitted from the staining
procedure. All antibodies were diluted in PBS containing
0.1% Triton X-100 and 1% normal goat serum.
Quantifications and data analysis
Quantitative analysis of immunopositive cells in the TG
and in the brainstem was performed by using the NIH
Image-J software on digital images of immunolabeled
sections. To avoid variability in the staining procedure,
all the sections to be compared were processed together,
and images were acquired under the same exposure
conditions. Two sections for each TG or brainstem and
for each labeling antibody were analyzed.
The number of Iba1+ macrophages in TG was measured on sections captured at 10× magnification. A
stack of all acquired images was created, and the
threshold for analysis was set to a level that included
all Iba1-immunopositive cells, but not the lighter pixels
of the background. The perineuronal regions of the V1,
V2 and V3 division were outlined, and both the number and the average size of Iba1+ cells were then measured. The number of positive cells has been
normalized to the outlined area of measurement. The
number of ED1+ macrophages in TG was estimated by
counting the number of immunopositive cells at 40×
magnification.
ED1 immunoreactivity in the TG, and GFAP, Iba1 and
P2Y12 receptor immunoreactivity in the brainstem was
assessed by densitometric analysis. A digital image of
the immunolabeled sections was acquired at 20× or 10×
magnification for TG or brainstem, respectively, and the
threshold for analysis of positive staining was set as
described above. The mean values of pixel intensity
were then automatically counted by using the NIH
Image-J software. For GFAP-, Iba1- and P2Y12- receptor
immunostaining, the mean pixel intensity values were
expressed as fold increase compared to the contralateral
side of saline injected animals set to 1.0. The anatomical
structures in the brainstem and spinal cord were identified with reference to a rat brain atlas [74].
All results are expressed as mean ± s.e.m. of at least
three independent experiments. Statistical significance
between groups was derived from one-way ANOVA followed by Scheffe’s analysis, performed with the SPSS
Villa et al. Molecular Pain 2010, 6:89
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software. Three degrees of significance were considered:
p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).
Abbreviations
CFA: Complete Freund’s Adjuvant; CNS: central nervous system; DRG: dorsal
root ganglia; Iba1: ionized calcium binding adaptor molecule 1; GFAP: glial
fibrillary acidic protein; IL: interleukin; PNS: peripheral nervous system; RT:
room temperature; SGCs: satellite glial cells; TG: trigeminal ganglion; TMJ:
temporomandibular joint; TNFa: tumor necrosis factor-a.
Acknowledgements
Authors are deeply thankful to Mrs. Loredana Bonacina for her skillful help in
managing and housing animals, and to Dr. Paolo Gelosa for his advices and
help.
The financial support of Telethon - Italy (Grant #GGP07032A entitled:
“Genetic and micro-environmental factors regulate the role of ATP as
transmitter of pain in a migraine model”, and grant #GGP10082A entitled:
“Studies of familial hemiplegic migraine transgenic mouse models and
patients to investigate the crosstalk between sensory neurons and
neuroinflammatory cells in trigeminal ganglia in relation to migraine pain”,
to MPA) is gratefully acknowledged.
Author details
Department of Pharmacological Sciences, Università degli Studi di Milano,
via Balzaretti 9, 20133 Milan, Italy. 2Department of Neurosurgery, Cedars Sinai
Medical Center, Los Angeles CA 90013, USA. 3Department of Anatomy,
University of California San Francisco, San Francisco, CA 94143, USA.
1
Authors’ contributions
GV contributed to the study design, performed all the experiments, acquired
and analyzed data and drafted the manuscript. MZ contributed to in vivo
studies and to the acquisition and analysis of data. GM contributed to in
vivo studies and to the acquisition and analysis of data
SC participated to the study design, supervised the experimental procedures,
and drafted the manuscript. LJ and PTO contributed to the study design
and drafted the manuscript. MPA participated to the study design,
supervised the experimental procedures and drafted the manuscript. All
authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 15 September 2010 Accepted: 10 December 2010
Published: 10 December 2010
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doi:10.1186/1744-8069-6-89
Cite this article as: Villa et al.: Temporomandibular joint inflammation
activates glial and immune cells in both the trigeminal ganglia and in
the spinal trigeminal nucleus. Molecular Pain 2010 6:89.
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