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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: J Pain. 2009 Jan 29;10(3):336–342. doi: 10.1016/j.jpain.2008.10.010

Acid sensing ion channel 3 expression in mice knee joint afferents and effects of carrageenan-induced arthritis

M Ikeuchi 1,2, SJ Kolker 1, KA Sluka 1
PMCID: PMC2675631  NIHMSID: NIHMS102569  PMID: 19185546

Abstract

Arthritis is associated with decreases in local pH. Of the acid sensing ion channels (ASIC), ASIC3 is most sensitive to such a pH change, abundantly expressed in dorsal root ganglion (DRG), and critical for the development of secondary hyperalgesia. The purpose of this study was to investigate the upregulation of ASIC3 using an acute arthritic pain model in mice. We examined ASIC3 expression in DRG neurons innervating the knee joint with and without carrageenan-induced arthritis by means of retrograde labeling and immunohistochemistry. We also examined the difference of DRG phenotype between ASIC3+/+ and ASIC3-/- mice. ASIC3 immunoreactivity was present in 31% of knee joint afferents and dominantly in small cells. After joint inflammation, ASIC3-immunoreactive neurons significantly increased in number by 50%. CGRP increased similarly in both ASIC3+/+ and ASIC3-/- mice. Soma size distribution of ASIC3-immunoreactive neurons without CGRP expression was shifted to smaller diameter neurons. Our results suggest that ASIC3 plays an important role in acute arthritic pain. Specifically, we propose that ASIC3 upregulation along with CGRP and phenotypic change in ASIC3-immunoreactive neurons without CGRP are responsible for the development of secondary hyperalgesia following carrageenan-induced arthritis.

Keywords: ASIC, joint, pain, DRG, inflammation, acid, CGRP

Perspective

This article shows that ASIC3 is upregulated along with CGRP in knee joint afferents and there is a phenotypic change in ASIC3-immunoreactive non-peptidergic neurons in an animal model of acute arthritis. Understanding the basic neurobiology after acute arthritis could lead to future new pharmacological management of arthritis.

Arthritis is currently one of the most frequent chronic health problems, particularly osteoarthritis. Clinical symptoms are dominated by chronic joint pain, which leads to disability, psychological distress, and impaired quality of life. Despite its frequency and impact, the mechanisms of joint pain are largely unknown3,19. Joint pain is uniquely different from cutaneous pain and characterized as diffuse, longer lasting and more unpleasant27,32. It is often accompanied by referred pain and secondary hyperalgesia27,37, i.e. increased nociceptive response to noxious stimuli outside the joint. This may relate to differences in different biochemical mediators or central anatomical pathways. Dorsal root ganglion (DRG) neurons innervating joint have more calcitonin gene-related peptide (CGRP) and substance P and less isolectin B4 (IB4) and somatostatin when compared to DRG neurons innervating the skin16,24. The central projections from nociceptors innervating joint tissue are predominantly to laminae I and deeper dorsal horn, while those from cutaneous tissue project to laminae II29. Although some important knowledge about joint pain is accumulating, that is not sufficient to develop more effective treatment for arthritic pain. In fact, nonsteroidal anti-inflammatory drugs and acetaminophen are still the class of medication most commonly used for arthritic pain while their effects are limited9,41.

At the site of arthritis, a number of inflammatory substances are released into the joint from nerves, immunocytes, synoviocytes, platelets, vascular endothelium, and interstitial fluid. Many of these substances are capable of activating free nerve endings of joint afferents, ultimately resulting in the perception of joint pain2,19. Protons are one of these inflammatory substances and can directly activate nociceptors by opening proton-gated cation channels, such as acid sensing ion channels (ASICs) and capsaicin sensitive TRPV12,5. Synovial fluid in inflamed knee joint shows a drop in pH to levels around 6.68. Among the proton–gated cation channels, ASIC3 is the most sensitive to such a pH change17,40, abundantly expressed in DRGs38, and strongly correlated with pain25,33,34,36. Although ASIC3 is considered to be one of the future targets to control joint pain5,30, there have been no studies about ASIC3 in joint afferents except our previous report13. Our laboratory recently showed that secondary hyperalgesia following joint inflammation (response to von-Frey filaments applied to the paw) does not develop in ASIC3 knockout (ASIC3-/-) mice while the primary mechanical hyperalgesia (response to tweezer applied to the inflamed knee joint) develops similarly between ASIC3-/- mice and wildtype mice (ASIC3+/+)13. Therefore, we concluded that ASIC3 is critical for the development of secondary hyperalgesia. In addition, we observed that ASIC3 immunoreactive (IR) peripheral nerves were present in inflamed, not in uninflamed, synovium of the knee joint, and that these ASIC3 positive fibers co-localized with CGRP13. We hypothesized that ASIC3 was present only in a small proportion of DRG neurons innervating the uninflamed knee joint and that carrageenan-induced arthritis resulted in ASIC3 upregulation that was responsible for the development of secondary hyperalgesia.

In this study, we evaluated the ASIC3 expression of DRG neurons innervating the knee joint and the effects of carrageenan-induced arthritis with particular reference to CGRP co-expression. We also examined the difference of DRG phenotype between ASIC3+/+ and ASIC3-/- mice in terms of soma size distribution and CGRP expression. Use of ASIC3-/- mice allows us to confirm specificity of the ASIC3 staining and examine the role of ASIC3 in DRG phenotype.

Materials and Methods

Animals

We used congenic ASIC3-/- mice and ASIC3+/+ (C57/Bl/6J) mice (age 2-3 months) (Jackson Laboratories, Bar Harbor, ME). The generation of the ASIC3-/- mouse is described in detail elsewhere26,39. All experiments were approved by the Animal Care and Use Committee at the University of Iowa.

Retrograde labeling of knee joint afferents

Animals were deeply anaesthetized with 2.5-5% isoflurane. After shaving, a 5-6mm long skin incision was made at the left knee joint and 0.1mg Fast Blue (FB) (Polysciences, Inc. Warrington, PA) diluted in 10 μl of saline was injected into the joint cavity for retrograde labeling. The wound was closed with 5-0 silk. FB containing neurons were identified in vitro by blue fluorescence emission on brief exposure of the cells to ultraviolet light.

Induction of joint inflammation

At 6 days after FB injection, an injection of 20 μl of 3% carrageenan (20 μl of saline for uninflamed control animals) was given into the left knee joint percutaneouly while the mice were briefly anaesthetized with 3% isofluorane. The carrageenan arthritis model has been extensively studied and it produces primary and secondary hyperalgesia to mechanical and heat stimuli, which develops rapidly within hours and persists for weeks after injection13,27,29.

Immunohistochemistry

At 7 days after FB injection, i.e. 24 hours after carrageenan injection, animals were euthanized with an overdose of sodium pentobarbital (150 mg/kg, i.p.) and the ipsilateral L3-L5 DRG were removed. The contralateral L3-L5 DRG from 3 mice were also removed to examine for systemic spread of FB. The DRGs were placed in 2% paraformaldehyde and 15% sucrose overnight, embedded in OCT compound (Sakura Finetek, Torrace, CA, USA), frozen on dry ice, and kept in -70°C until sectioning. Ten-micrometer frozen sections were then cut using a cryostat.

For simultaneous visualization of ASIC3 with CGRP, a double immunofluorescence method was used. The primary antisera used in this study were rabbit anti-ASIC3 serum (1:500; Alomone Labs, Jerusalem, Israel) and rabbit anti-CGRP serum (1:1000; Peninsula Laboratories, SanCalros, CA). For visualization of ASIC3 antibody, avidin-biotin complex method was used. Because anti-ASIC3 and anti-CGRP serum were raised in a same species (rabbit), nonspecific rabbit IgG and goat anti-rabbit monovalent Fab fragment were used between ASIC3 and CGRP staining in order to avoid cross-reactivity between the detection systems. Absence of cross-reactivity was confirmed by omitting either of the primary antibodies.

The sections were blocked in 3% normal goat serum for 30 minutes, then incubated in rabbit anti-ASIC3 serum overnight in a humid atmosphere. The next day, the sections were incubated with goat anti-rabbit biotinylated IgG (1:250; Vector, Burlingame, CA) for 2 hours followed by strepavidin Alexa 568 (1:500; Invitrogen, Carlsbad, CA) for 2 hours. Subsequently, the sections were incubated with nonspecific rabbit IgG (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour to absorb the excess goat anti-rabbit biotinylated IgG, followed by monovalent Fab fragment (final concentration 0.025 mg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour to absorb the excess nonspecific rabbit IgG. Afterwards, the sections were incubated with rabbit anti-CGRP serum overnight in a humid atmosphere. On the 3rd day, the sections were incubated with goat anti-rabbit Alexa 647 (1:500; Invitrogen, Carlsbad, CA) for 2 hours. All antisera used were diluted in PBS containing 1% normal goat serum and 0.05% Triton X-100. Before, between, and after each incubation step, the sections were washed with 5 times for 5 minutes in PBS. Finally, all sections were mounted with Vectashield (Vector, Burlingame, CA).

Microscopic observation

Sections were viewed with Olympus BX-51 microscope (Olympus, Tokyo, Japan). Representative photos were taken with Bio-Rad Radience 2100MP Multiphoton/Confocal Microscope (Bio-Rad, Richmond, CA). We counted FB-labeled neurons with visible nuclei from every fifth section to eliminate the possibility of double counting. More than 100 FB-labeled neurons were analyzed from 4 mice in each group. For each FB-labeled neuron, ASIC3 and CGRP expression was examined and quantified as the percent of total FB-labeled neurons. Data was presented as the mean (%) ± SEM. Soma size of FB-labeled neurons was measured using imaging software as the area of the cell in μm2 (Image J; National Institutes of Health, available at: http://rsb.info.nih.gov/ij/). Soma size distribution was calculated as the total for each population, retrogradely labeled knee joint afferents, CCRP-IR afferents, and ASIC3-IR afferents and so on, and divided into 6 different categories: <250, <500, <750, <1000, <1250, <1500, as previously described28.

Statistical analysis

Student's t-test was used for between-group (uninflamed vs inflamed) comparisons in ASIC3-/- mice. For comparisons in the number of knee joint afferents and CGRP-IR cells, two way-analysis of variance (ANOVA) followed by Tukey's test was used. For the evaluation of soma size distribution, Kolmogorov-Smirnov test was used to determine whether the distribution of soma size differed between each population. The level of significance was set at p<0.05.

Results

Knee joint afferents in ASIC3 +/+ and ASIC3 -/- mice

Intense FB labeling was found in DRG somata without leakage of fluorescence into the surrounding tissue (Fig. 1A, 1B, 1C, 1D). Twenty to thirty eight FB-labeled neurons were observed from L3-L5 DRG in each animal. L4 DRG usually contained most FB-labeled neurons among L3-L5 DRG. No significant difference was seen among the number of FB-labeled neurons of each of the 4 groups: ASIC3+/+ and ASIC3-/- with and without inflammation (Fig. 2A). Fig. 3A shows a histogram of the soma size distribution of FB-labeled neurons. There was a unimodal distribution with a broad range of sizes. The median soma size of FB-labeled neurons was 425 μm2 in ASIC3+/+ and 413 μm2 in ASIC3-/- mice. No significant difference was observed among the soma size distribution of FB-labeled neurons of each group. There was no non-specific systemic spread of FB since the contralateral DRGs had no FB staining.

Fig. 1.

Fig. 1

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Fast Blue labeling (A,B,C and D) and immnohistochemistry staining for ASIC3 (E,F,G and H) and CGRP (I,J,K and L) showing DRG from ASIC3+/+ (first and second rows) and ASIC3-/- (third and fourth rows) mice without joint inflammation (first and third rows) and those 24h after joint inflammation (second and fourth rows). Photos in each row are the same DRG. Arrows indicate Fast Blue labeled DRG neurons. Asterisks indicate ASIC3-IR neurons. No immunoreactivity for ASIC3 was observed in ASIC3-/-mice(G and H), while CGRP was observed in all groups. Some DRG neurons showing both ASIC3 and CGRP immunoreactivity were observed as yellow in ASIC3+CGRP merged images (M and N).

Fig. 2.

Fig. 2

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The number of knee joint afferents (A) and the percentage of knee joint afferents immunoreactive for CGRP (B), ASIC3 (C), ASIC3+CGRP (D) and ASIC3 without CGRP (E). Values are represented as mean±SEM. Knee joint afferents immunoreactive for CGRP, ASIC3 and ASIC3+CGRP were significantly increased 24h after carrageenan-induced arthritis. *p<0.05 vs. uninflamed mice.

Fig. 3.

Fig. 3

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Soma size distribution of knee joint afferents (A) was unimodal with a broad range of size and similar to that of ASIC3-IR knee joint afferents (C). CGRP-IR knee joint afferents consisted of small to medium cells with most cells smaller than 500μm2 (B). ASIC3 and CGRP double labeled knee joint afferents were small to medium as well (D) whereas ASIC3 without CGRP-IR knee joint afferents were larger (E). There were no significant differences in soma size distribution among each group in knee joint afferents, CGRP, ASIC3 and ASIC3+CGRP. Only significant difference in soma size distribution was observed in ASIC3 without CGRP (E). Carrageenan-induced arthritis resulted in an overall shift in soma size distribution of ASIC3 without CGRP-IR knee joint afferents to the left (E). *p<0.05 between uninflamed vs. inflamed mice.

ASIC3 and CGRP expression in uninflamed knee joint afferents

DRGs contained many ASIC3-IR and CGRP-IR neurons (Fig. 1). No immunoreactivity for ASIC3 was observed in ASIC3-/- mice, while CGRP was observed in all groups. Co-localization of ASIC3 with CGRP was frequently observed in DRG. In normal uninflamed animals, counts revealed that 31 ± 5 % of knee joint afferents were ASIC3-IR in ASIC3+/+ mice. Similar amounts of CGRP were observed in ASIC3 +/+ and ASIC3 -/- mice without inflammation: 29 ± 3 % (ASIC3+/+) and 32 ± 1 % (ASIC3-/-). The percentage of FB-labeled neurons that expressed both ASIC3 and CGRP was 18 ± 2 %, and the percentage of FB-labeled neurons that expressed ASIC3 without CGRP was 14 ± 3 % (Fig. 2). CGRP was found in 62 ± 11 % of ASIC3-IR neurons retrogradely labeled from the knee joint, and 94 ± 5 % of ASIC3-IR small neurons (<500 μm2). Although more than half (55 %) of ASIC3-IR neurons were smaller than 500 μm2, their soma size distribution was unimodal with a broad range of size (median 419 μm2). CGRP-IR neurons consisted of small to medium cells with most cells smaller than 500 μm2 (median 296 μm2 in ASIC3 +/+ and 395 μm2 in ASIC3 -/-). Neurons that showed co-localization of ASIC3 with CGRP were small to medium size (median 259μm2) whereas ASIC3 neurons that did not co-localize with CGRP were larger (median 725μm2). There was no significant difference in number and soma size distribution of knee joint afferents and CGRP-IR neurons between ASIC3+/+ and ASIC3-/- mice (Fig. 3).

Effects of carrageenan-induced arthritis

All mice injected with carrageenan showed swelling of the knee joint and abnormal gait posture with holding up the affected leg 24 h after carrageenan injection. Counts revealed that knee joint inflammation significantly increased the number of CGRP-IR neurons retrogradely labeled from the knee joint from 29 ± 3 % to 48 ± 1 % in ASIC3+/+ and from 32 ± 1 % to 51 ± 5 % in ASIC3-/- mice (F1,15=40.7, p=0.0001). ASIC3-IR neurons retrogradely labeled from the knee joint significantly increased in number from 31 ± 5 % to 47 ± 2 % (p=0.019). Further, there was a significant increase, from 18 ± 2 % to 31 ± 4 %, in the number of neurons that showed co-localization of ASIC3 with CGRP in knee joint afferents 24h after induction of inflammation (p=0.034) (Fig. 2). The increase in the number of CGRP-IR neurons after inflammation was not significantly different between ASIC3+/+ and ASIC3-/- mice; and there was no significant difference in the number of ASIC3-IR neurons without CGRP expression between uninflamed and inflamed mice.

Soma size distribution after inflammation was similar to that of uninflamed mice in knee joint afferents for CGRP, ASIC3, and co-localization of ASIC3 with CGRP. However there was a significant difference in the soma size distribution between uninflamed and inflamed mice in the ASIC3-IR neurons that did not express CGRP (p=0.027) (Fig. 3). The median soma size of ASIC3-IR neurons without CGRP expression decreased from 725 μm2 in uninflamed to 572 μm2 in inflamed mice, suggesting there was an upregulation of ASIC3 in smaller non-peptidergic neurons.

Discussion

ASIC3 subpopulation in uninflamed knee joint afferents

Our study showed that ASIC3 expression was observed in 31% of uninflamed knee joint afferents in ASIC3+/+ mice. There was no ASIC3 expression in ASIC3-/- mice confirming the specificity of the antibody to ASIC3. In our previous study, however, we did not see ASIC3 expression in peripheral nerve fibers in uninflamed synovium of the mice knee joint13. One possible reason for the difference in ASIC3 expression between DRG and peripheral terminals could be a greater localization of ASIC3 in the cell bodies of DRG neurons. Garcia-Anoveros et al.7 reported that ASIC2a is actively transported from soma to peripheral terminals. It is possible, however, that ASIC3 may not be transported to peripheral terminals in the synovium of the knee joint sufficient quantity to be detected by means of immunohistochemistry, especially in the uninflamed knee joint. In other words, ASIC3 transport to the peripheral terminals innervating the knee joint may occur only after inflammation. Further studies involving nerve transaction are necessary to confirm changes in ASIC3 transport in the axonal flow during acute arthritis. Another possible reason is the difference in background staining between joint tissue and DRG. The synovium is immediately adjacent to fat which results in a greater background fluorescence using the avidin-biotin amplification technique.

ASIC3 was present in diverse DRG neurons in terms of soma size. Although the histogram showed small cells (<500 μm2) were dominant in ASIC3-IR population (55 %), there was considerable number of ASIC3-IR middle to large cells (>500 μm2). This finding is consistent with previous reports about ASIC3 population in rat sensory neurons12,20. Because nociceptors generally belong to small to medium size neurons11, it seems reasonable to suppose that ASCI3 is present in both nociceptors and non-nociceptors among knee joint afferents.

ASIC3 expression is different among specific tissue afferents12,20. Ichikawa et al.12 reported that there was more ASIC3 expression in tooth pulp afferents than skin afferents in rats (33% vs 13%), and also that co-localization of ASIC3 with CGRP in tooth pulp afferents was more frequent than that in skin (100% vs 81%). Molliver et al.20 reported similar results comparing muscle afferents with skin afferents in rats. There was more ASIC3 expression (50% vs 28%) and co-localization of ASIC3 with CGRP in muscle afferents than skin afferents (83% vs 69%). In the current study, ASIC3 was present predominantly in peptidergeic neurons among small cell population because most of ASIC3-IR small cells (94%) co-expressed CGRP. This is consistent with prior reports4,18 showing IB4-negative small neurons, i.e. peptidergic neurons, are the primary responders to protons with a higher prevalence and greater amplitude of ASIC currents compared to IB4-positive small neurons, i.e. non-peptidergic neurons. An increased response to protons through ASIC3 in peptidergic neurons may well be due to unique characteristics of tooth pulp afferents and deep tissue afferents.

Effects of joint inflammation

Our study showed that there was an upregulation of both ASIC3 (50%) and CGRP (64% in ASIC3+/+ and 57% in ASIC3-/-) by carrageenan-induced arthritis. Although there are a few reports of ASIC3 upregulation by inflammation in rat DRG at the mRNA38 and protein level25, this is, to our knowledge, the first report about ASIC3 upregulation by inflammation specifically in knee joint afferents. The upregulation of CGRP observed in the current study after inflammation in mice is consistent with previous reports specific to knee joint afferents in cat10 and rat6. In terms of soma size distribution, not only CGRP but also ASIC3 was present predominantly in small to medium cells, i.e. putative nociceptors, after joint inflammation. Therefore, ASIC3 upregulation presumably contributes to development of arthritic pain, as CGRP does6,10,23,35.

We found a significant change of soma size distribution for ASIC3 in neurons that do not express CGRP after joint inflammation. Carrageenan-induced arthritis resulted in an overall shift in its distribution to the left (smaller). Meanwhile, there was no significant change in soma size distribution for ASIC3 in peptidergic neurons. Although the immunohistochemical absence of CGRP does not necessarily mean non-peptidergic, these data presumably suggest a relatively increased expression of ASIC3 in non-peptidergic small neurons21.

Role of ASIC3 in phenotype of knee joint afferents

This study was the first to examine ASIC3 distribution in DRG neurons innervating the knee joint in mice, and to confirm this distribution using ASIC3-/- mice. We also examined the phenotype of DRG neurons using both ASIC3+/+ and ASIC3-/- mice. CGRP was used to label peptidergic neurons to examine distribution between peptidergic and non-peptidergic neurons. Although IB4 is a commonly used marker for non-peptidergic neurons, IB4 was not used because there are few IB4 binding neurons in joint afferents14,22.

Interestingly, there was no difference between ASIC3+/+ and ASIC3-/- mice in every comparison except ASIC3 expresssion, including the number, soma size distribution, and effects of inflammation. CGRP upregulation was the only change similarly observed in both ASIC3+/+ and ASIC3-/- mice following joint inflammation. Meanwhile, significant differences between ASIC3+/+ and ASIC3-/- mice with arthritis showed an upregulation of ASIC3 and ASIC3 in CGRP positive neurons, as well as a phenotypic change in ASIC3 neurons without CGRP.

In terms of pain behaviors, our previous study13 showed that secondary hyperalgesia did not develop in ASIC3-/- mice while primary hyperalgesia of the inflamed knee joint developed in ASIC3-/- mice and was similar to ASIC3+/+ mice. Previous data similarly show that secondary mechanical hyperalgesia does not develop after muscle insult33,34. Taken together, these data suggest that the upregulation of ASIC3 in CGRP positive neurons, and a phenotypic change in neurons expressing ASIC3 without CGRP are responsible for the secondary hyperalgesia.

It is unknown why ASIC3 plays such a significant role in the development of secondary, but not primary, hyperalgesia. Secondary hyperalgesia is widely accepted to result from sensitization of dorsal horn neurons31, and characterized by enhanced nociception to mechanical stimuli1. There is rarely a direct monosynaptic pathway from nociceptive primary afferents to dorsal horn neurons15. As such, Ziegler et al.42 proposed that central sensitization of nociceptive pathways involves a more complex circuitry than is assumed by a single-neuron model. Further, central sensitization does not occur after muscle insult in ASIC3 -/- mice33. If central sensitization is driven mainly by proton-sensitive afferents innervating the inflamed knee joint, ASIC3 upregulation in knee joint afferents could induce central sensitization that is independent of primary mechanical hyperalgesia of the inflamed knee joint.

In conclusion, our results suggest that ASIC3 in knee joint afferents plays an important role in acute arthritic pain. Specifically, ASIC3 upregulation along with CGRP and the phenotypic change of ASIC3-immunoreactive neurons without CGRP are hypothesized to be responsible for the development of secondary hyperalgesia following carrageenan-induced arthritis.

Acknowledgments

Supported by the National Institutes of Health AR053509.

Footnotes

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