Intraplantar inflammation
Previous studies found that on intraplantar inflammation,
ASIC3-/- mice exhibited either no or slightly enhanced hyperalgesia [
14,
15]. Although mouse strains and sexes of these two studies may cause the discrepant results from our current work, the studies used 2% carrageenan, and the tests were carried out only 3–4 h after injection. In another study involving dominant-negative
ASIC3 transgenic mice [
21], behavior tests were performed 1–6 h after zymosan injections to induce paw inflammation. These mice exhibited increased mechanical hypersensitivity. Although the role of ASIC3 in the acute phase of inflammation is inconclusive, our results indicate the involvement of ASIC3 in the sub-acute phase of inflammation. Our study showed no difference in nociceptive behaviors in the acute phase of inflammation (4 h) between two genotypes, except for a small but significant difference in thermal hyperalgesia in the carrageenan inflammatory model. On follow-up in
ASIC3-/- mice, in both models for mechanical hyperalgesia and in the carrageenan model for thermal hyperalgesia, hyperalgesia was attenuated after intraplantar injection. The inflammation state might have changed from an acute to a sub-acute phase in 24 h, and
ASIC3-/- mice could have recovered faster from hyperalgesia. Although ASIC3 is believed to play a more important role in secondary hyperalgesia (hypersensitivity occurred in non-injured sites) induced by inflamed muscle or joints than primary hyperalgesia in inflamed tissues [
16,
17], in this study we provide evidence that ASIC3 does play a role in subcutaneous primary hyperalgesia in later stages. This conclusion is also supported by a recent report showing that subcutaneous injection of the ASIC3-selective blocker APETx2 can alleviate the inflammation-induced hyperalgesia [
22]. However, the role of ASIC3 for sub-acute-phase primary hyperalgesia is not applied to the deep tissues, because a recent study reported that
ASIC3-/- mice did not show different levels of primary hyperalgesia in inflamed joint induced by 3% of carrageenan than
ASIC3+/+ mice [
17].
We did not observe a significant difference in effects of inflammation between ASIC3+/+ and ASIC3-/- mice on pathological examination, which indicates that ASIC3 does not play a role in the development of inflammation, and the difference in hyperalgesia may not be related to the degree of inflammation.
During inflammation, the enhanced expression of ion channels sensitizes primary afferent neurons and produces hyperexcitability, thereby producing hyperalgesia. Up-regulation of ASIC3 and sodium channels during inflammation have been documented [
8‐
10,
23‐
25]. However, real-time PCR results in our study showed no transcriptional change in
ASIC3,
Nav1.6,
Nav1.7, and
Nav1.8 mRNA level following inflammation. These results are contradictory to those in previous studies. The only up-regulated gene we found was
Nav1.9, an ion channel reported to be unchanged under intraplantar carrageenan-induced inflammation [
25].
Nav1.9 mRNA level has been shown to increase by day 7 with CFA-induced inflammation [
26]; however, its expression was not changed in our CFA model. A possible explanation for the discrepancy between our study and previous studies is the difference in sampling and signal quantification. Previous studies used methods such as RT-PCR,
in situ hybridization or immunostaining, which were semi-quantitative. Furthermore, both
in situ hybridization and immunostaining examine one plane of cells, whereas with real-time PCR, the total mRNAs from all cellular populations in a single DRG were quantified. Lack of transcriptional change in a single DRG by real-time PCR cannot rule out the possibility of up- or down-regulated genes in subgroups of cells or involvement of the gene in posttranscriptional regulation for the process of sensitization. Another issue to consider is the timing of sampling, since regulation of these genes might be transient and time dependent. Animal species and genetic background may also account for the discrepancies to a certain extent, because mice express relatively less ASIC channels in DRGs than do rats [
27,
28].
The up-regulation of
Nav1.9 on day 2 with intraplantar carrageenan-induced inflammation was significant and ASIC3 dependent. The functional importance of
Nav1.9 in modulation of pain behavior in inflammation has been previously investigated by disrupting the ion channel in mice [
29]. Mechanical and thermal thresholds are comparable between
Nav1.9-/- and wild-type mice in the absence of injury. In contrast, inflammation-mediated pain behavior differs prominently in
Nav1.9-/- mice as compared with wild-type mice. Intraplantar injection of carrageenan induced thermal hyperalgesia in both wild-type and
Nav1.9-/- mice in the first 3 h post-injection; however, the hyperalgesia was diminished 24 h later in
Nav1.9-/- mice. This finding matches our observation of
ASIC3-/- mice with longer paw withdrawal latency starting at 4 h and continuing through day 2, which is indicative of attenuated thermal hyperalgesia.
The similar phenotypes in the pain behavior for Nav1.9-/- and ASIC3-/- mice under inflammation and the ASIC3-dependent up-regulation of Nav1.9 suggest that inflammation induces tissue acidosis, which leads to activation of ASIC3 and subsequent ASIC3-dependent up-regulation of Nav1.9; the increased Nav1.9 level in turn contributes to thermal hyperalgesia. In the absence of ASIC3, Nav1.9 cannot be up-regulated, and thermal hyperalgesia becomes attenuated.
In contrast to up-regulation of
Nav1.9 transcripts, increase of I
Nav1.8 on day 2 with intraplantar carrageenan-induced inflammation was significant and ASIC3 dependent on electrophysiology (Fig.
11). Although our electrophysiolgical data did not show increased I
Nav1.9 in overall small to medium neurons to support the up-regulation of
Nav1.9 transcripts during inflammation, we did find a significant increase of I
Nav1.9 in neurons that did not express Nav1.8 activity in
ASIC3+/+ mice (65.4 ± 16.0 vs. 506.5 ± 147.7 pA for the control vs. carrageenan group, respectively, P < 0.05). Further study would aim to determine whether a specific subset of DRG neurons plays an important role in regulating the ASIC3-dependent effect during inflammation. However, the increase in I
Nav1.8 was robust, with an increase of up to threefold in peak amplitudes, which was associated with maximal mechanical and thermal hyperalgesia on day 2 of carrageenan inflammation. However, a previous study reported slight change of I
Nav1.8 peak amplitude on day 4 of carrageenan inflammation, when the
Nav1.8 mRNA was significantly up-regulated and the current density of I
Nav1.8 was slightly increased [
24]. Thus, the alteration of Nav1.8 activity during inflammation may be time-dependent, as was seen in many studies [
25,
30]. Nevertheless, the increase of I
Nav1.8 is intriguing because Nav1.8 is known to play a role in mechanical nociception, and inhibition of Nav1.8 would abolish inflammation-induced mechanical and thermal hyperalgesia [
31,
32]. Therefore, the increase in Nav1.8 activity may account in part for the carrageenan-induced mechanical hyperalgesia found in
ASIC3+/+ mice but not in
ASIC3-/- mice.
Intramuscular inflammation
ASIC3 is critical for the development of secondary mechanical hyperalgesia with muscle inflammation [
16]. Our findings agree with this result and show
ASIC3+/+ mice with significant mechanical hyperalgesia 2 days after intramuscular carrageenan injection. However, we did not observe thermal hyperalgesia in either genotype with carrageenan-induced inflammation as was reported for C57BL6 mice [
16]. Perhaps the time to establish secondary thermal hyperalgesia in CD1 mice is longer than is needed in C57BL6 mice.
In inflamed muscle,
ASIC3-/- mice seemed to display milder pathological features, including infiltration of leukocytes, formation of granulomas and vasculitis, than
ASIC3+/+ mice. Previous reports described the same pathological responses in both genotypes, and an assessment of neutrophilic activity by myeloperoxidase in carrageenan-inflamed muscle also suggested that ASIC3 played no role in the development of inflammation [
16]. Although neutrophilic activity represents only one aspect of inflammation, the type of immune cells recruited and their organization and behavior vary by inflammatory conditions. As we examined closely, the inflammation processes involved in CFA- or carrageenan-induced inflammation are not simple. Carrageenan-induced vasculitis was not documented, although in one study histological examination was carried out to characterize the transition of immune cell-type from acute to chronic inflammation of the rat leg muscle [
33]. Also, CFA has not been used as a muscle inflammation model; thus, no report of its effect on granuloma formation in muscle exists.
The development of granulomas and vasculitis involves complicated interaction among immune cells, cytokines and chemokines. Granulomas are the aggregation of mononuclear inflammatory cells and modified macrophages organized in a compact nodule, with involvement by a complicated interaction between antigen-presenting macrophages and T lymphocytes mediated by various cytokines [
34]. However, the pathogenesis of vasculitis is still not well understood, but endothelium injury is generally believed to be the fundamental event in its development. Responding to neuropeptides (substance P or calcitonin gene-related peptide), endothelial cells mediate several features of chronic inflammation such as vasodilatation, leukocyte migration, cytokine production and cellular adhesion molecule expression [
35]. During vascular tone regulation and interaction with immune cells, damage to endothelial cells could occur [
36].
The involvement of ASIC3, an ion channel present on neurons, in the pathogenesis of these features is thus intriguing. The interplay between the neuronal and immune system has become an interesting topic recently; body systems do not work alone. Neurons could initiate a cascade of cytokine synthesis and release and recruitment of inflammatory machinery. The process is neurogenic inflammation, whereby small-diameter sensory neurons release neuropeptides such as substance P and calcitonin-gene related peptide upon activation. Blocking nerves results in decreasing the inflammation consequences of carrageenan-induced inflammation [
37]. ASIC3 could thus participate in the pathogenesis of granuloma formation and vasculitis through activating neurogenic inflammation. Lacking ASIC3 may alter the properties of sensory neurons [
38], thus influencing its neurogenic release and the inflammatory consequences. This point should be further investigated. However, the immune system could also affect how the neural system perceives pain through activated mast cells, macrophages, neutrophils, and the cytokines they release [
1]. ASIC3-mediated inflammation could be essential for the hyperalgesia we observed in wild-type mice.