1. Introduction
The acid sensing ion channel 3 (ASIC3) is critical for the development of hyperalgesia to mechanical stimulation of the paw following muscle insult. Prior studies from our laboratory show that in ASIC3 −/− mice, mechanical hyperalgesia of the paw does not develop after repeated intramuscular injections of acid or after intramuscular inflammation induced by carrageenan [22,23]. Heat hyperalgesia of the paw still develops similarly between ASIC3 −/− and ASIC3 +/+ mice after carrageenan inflammation of the muscle [23]. These studies all measured an increased nociceptive response to noxious stimuli outside the site of injury, termed secondary hyperalgesia. Prior studies measuring mechanical and heat sensitivity of the paw after inflammation of the paw, i.e. primary hyperalgesia, show that hyperalgesia still develops in ASIC3 −/− mice and is either similar or increased to that of ASIC3 +/+ mice [1,7,13]. We previously interpreted this as a difference between cutaneous and muscle pain models. However, one could alternatively suggest that ASIC3 plays a different role in primary and secondary hyperalgesia.
Joint inflammation is associated with both primary hyperalgesia and secondary hyperalgesia. In an animal model of joint inflammation, induced by injection of kaolin and/or carrageenan into the knee joint, both primary and secondary hyperalgesia to mechanical stimuli develop [14,21,28]. Secondary hyperalgesia develops at the paw to heat and mechanical stimuli [14,24], and primary hyperalgesia is present over the inflamed knee joint [21]. This carrageenan-induced knee joint inflammation model produces sensitization of nociceptors innervating the knee joint, a substrate for primary hyperalgesia; and sensitization of neurons in the dorsal horn, a substrate for secondary hyperalgesia [2,11,16–18]. Well-characterized biochemical changes occur in the inflamed knee joint, the primary afferent fibers innervating the knee joint, and in the central nervous system including the spinal cord [19,20]. These biochemical changes may underlie the development and maintenance of inflammatory hyperalgesia, both primary and secondary.
ASIC3 is located in primary afferent fibers innervating the skin and the muscle [8,13,22]. It is unclear if ASIC3 is also located in primary afferent fibers innervating the joint. The localization of ASIC3 to primary afferent fibers innervating the muscle is critical to the development of secondary mechanical hyperalgesia after muscle insult [23]. We show that re-expression of ASIC3 in primary afferent fibers innervating muscle in ASIC3 −/− mice restores the secondary mechanical hyperalgesia of the paw that occurs after muscle inflammation [23]. Re-expression of ASIC3 in primary afferent fibers innervating the skin of ASIC3 −/− mice, however, has no effect on the mechanical hyperalgesia of the paw after muscle inflammation [23].
The purpose of this study was, therefore, to test the role of ASIC3 in the mechanical hyperalgesia associated with joint inflammation and to assess if ASIC3 is located in primary afferent fibers innervating the knee joint. We hypothesized that ASIC3 was necessary for both primary and secondary mechanical hyperalgesia that develops after joint inflammation. We also hypothesized that ASIC3 was located in primary afferent fibers innervating the knee joint.
2. Methods
2.1. Mice
All experiments were approved by the University of Iowa Animal Care and Use Committee and were conducted in accordance with National Institutes of Health guidelines. Congenic ASIC3 −/− and ASIC3 +/+ mice on a C57Bl/6J background were bred at the University of Iowa Animal Care Facility [13]. Both male and female mice, 4–8 months of age, were used in these studies. For behavior testing, mice were divided into ASIC3 +/+ (n=23) and ASIC3 −/− (n=24) groups. For immunohistochemical staining we tested tissue sections obtained from both ASIC3 +/+ (n=6) and ASIC3 −/− mice (n=6).
2.2. Behavior experiments with mechanical, thermal and joint sensitivity
2.2.1. Acclimation training
Mice were trained for 2 days on the mechanical plate, thermal plate and tweezer restraining glove. Separate groups of mice were tested for mechanical and thermal sensitivity of the paw and for mechanical joint sensitivity. On day 1, training consisted of placing the mice individually into ventilated plexiglass cubicles (9×3×5) on top of a wire-mesh table for 30min. This allowed the mice to acclimate to the confined area and to standing on the wire-mesh surface. Mice were then acclimated to thermal testing by placing them onto a flat glass surface in the same individual plexiglass cubicles, allowing the mice to get acclimated to the confined area and standing on a glass surface. In order to acclimate the mice to the tweezer regimen, mice were individually placed in a glove with their hind quarters outside of the glove and allowed to remain in this position for 5min per session. The mice were stroked to relax and had their hind paws gently pulled to acclimate them to being handled for tweezer testing. This was done twice a day for 2 days.
2.2.2. Mechanical testing of the paw
Mechanical sensitivity was tested using the von-Frey filaments for measuring cutaneous sensitivity. A von-Frey filament of bending force 0.04g was applied to the hind paws five times per trial and was repeated 10 times. An average of the 10 trials was used to generate one number at each testing time. A withdrawal was defined as positive if the mouse lifted its paw after the filament was pressed against the paw. Mechanical sensitivity was tested as follows: before injection of the knee joint (see Section 2.3), 24h after the injection, 72h after the injection, 1 week after the injection and 2 weeks after the injection. An increased number of responses was interpreted as secondary mechanical hyperalgesia.
2.2.3. Thermal testing of the paw
Thermal sensitivity was tested using a radiant heat source shone on the hindpaw of the mice until a withdrawal was produced. The latency to withdraw was measured as the paw withdrawal latency. The light source was applied to the hindpaws one time per trial and was repeated three times. An average of the three trials was used to generate one number per testing time. There were five testing periods: before injection of the knee joint (see Section 2.3), 24h after injection, 72h after injection, 1 week after injection and 2 weeks after injection. A decrease in latency was interpreted as heat hyperalgesia.
2.2.4. Mechanical testing of the joint
To measure joint sensitivity, the mouse was placed in a glove, each hindlimb extended and a pair of forceps applied to the knee joint until the animal withdrew from the stimulus. Three trials of squeezing the knee joint were applied at each testing period. The computer program TestPoint was used to measure the magnitude of each squeeze in millinewtons (mN). There were five testing periods: before injection of the knee joint (see Section 2.3), 24h after injection, 72h after injection, 1 week after injection and 2 weeks after injection. A decrease in threshold was interpreted as primary mechanical hyperalgesia.
2.3. Induction of inflammation
To induce knee-joint inflammation, an injection of 20μl of 3% carrageenan was injected into the left knee-joint cavity while the mice were briefly anaesthetized with isoflurane (3%).
2.4. Histology
For immunohistochemical staining two different protocols for tissue collection were used. All inflamed tissues were removed 24h after injection of 3% carrageenan into the knee joint. Figs. 2A, B, D, E, 3 and 4 were obtained by transcardially perfusing the mice with heparinized saline followed by 4% paraformaldehyde and removing the whole knee joint, including muscle and bone. Whole specimens were then decalcified for 4 weeks with 5% EDTA (ethylenediaminetetraacetic acid) with five changes of fresh solution per week. After complete demineralization, the tissues were rinsed thoroughly in phosphate buffered saline (PBS) and placed in 30% sucrose overnight. All tissues were rapidly frozen and stored at −80°C until being cut on a cryostat. Sections were then cryosectioned at 14μm and placed on slides using CryoJane Tape Transfer System (Instrumedics Inc., St. Louis, MO) for subsequent processing. The 20-μm cryosection in Fig. 2C was obtained by removal of the anterior synovial tissue, snap freezing at −160°C, and postfixing on the slide with 2% paraformaldehyde.
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Fig. 2:
(A) Sagittal-section through the knee joint of a mouse without inflammation stained with H&E depicting the location of the synovium. (B) High power magnification of the synovium of the knee joint showing synoviocytes lining the joint cavity and underlying fat cells. (C) Double label for PGP 9.5 (green, arrows) and ASIC3 (red), showing the nerve fibers innervating the synovium and ASIC3 localization in the synovium. (D) Shows ASIC3 localization in synovioctyes in ASIC3 +/+ mice. (E) Shows lack of ASIC3 staining in synoviocytes in ASIC3 −/− mice.
Fig. 3:
Immunohistochemical staining of the synovium from ASIC3 +/+ mice with joint inflammation stained for ASIC3 (A), PGP 9.5 (B). The merged image shows co-localization of ASIC3 and PGP 9.5 (C) depicted by arrows. Some PGP 9.5-labeled nerve fibers in the synovium do not localize with ASIC3 (arrowhead). Immunohistochemical staining from ASIC3 −/− mice with joint inflammation stained for ASIC3 (D), PGP 9.5 (E). There is no staining for ASIC3 in the ASIC3 −/− mice. The merged image shows no co-localization of ASIC3 and PGP 9.5 (F; arrowheads). Bar = 25 μm.
Fig. 4:
Immunohistochemical staining for ASIC3 and CGRP from sections in whole specimens showing the synovium from ASIC3 +/+ and ASIC3 −/− mice without joint inflammation and those 24 h after joint inflammation. (A–F) Immunoreactivity in animals without joint inflammation and (G–L) immunoreactivity in animals with joint inflammation. ASIC3 staining is shown in (A, D, G and J); CGRP staining is shown in (B, E, H and K); and the merged images of ASIC3 and CGRP are shown in (C, F, I and L). CGRP staining occurs in all groups, both ASIC3 +/+ and ASIC3 −/−. No immunoreactivity for ASIC3 was observed in ASIC3 −/− mice; and nerve fibers did not stain for ASIC3 in the ASIC3 +/+ mice. After joint inflammation, ASIC3 was located in nerve fibers innervating the synovium in ASIC3 +/+ mice. Some of these fibers co-localize and CGRP (arrows). However, some fibers stain positive for CGRP and do not localize with ASIC3 (arrowheads), and some fibers stain positive for ASIC3 that do not localize with CGRP (not shown).
For simultaneous visualization of ASIC3 with CGRP or PGP 9.5, a double immunofluorescence method was used. Preliminary experiments were carried out to determine the optimal dilution and the order of application of the primary antisera. The primary antisera used in this study were rabbit anti-ASIC3 serum (1:500; Alomone Laboratories, Jerusalem, Israel), rabbit anti-CGRP serum (1:1000; Peninsula Laboratories, SanCarlos, CA), and guinea pig anti-PGP 9.5 serum (1:1000; Neuromics, Minneapolis, MN). Because anti-ASIC3 and anti-CGRP serum were raised in the same species (rabbit), nonspecific rabbit IgG and monovalent Fab fragment of goat anti-rabbit were used between the ASIC3 and CGRP staining in order to avoid crossreactivity between the antibodies. The avoidance of crossreactivity was confirmed by omitting either of the primary antibodies.
The sections were blocked in 3% normal goat serum for 30min, then incubated in rabbit anti-ASIC3 serum overnight in a humid atmosphere. The next day, the sections were incubated with biotinylated goat anti-rabbit IgG (1:250; Invitrogen) for 1h followed by streptavidin Alexa 568 (1:500; Invitrogen, Carlsbad, CA) for 1h. Subsequently, the sections for PGP 9.5 labeling were incubated with guinea pig anti-PGP 9.5 serum overnight in a humid atmosphere. The sections for CGRP labeling were incubated with nonspecific rabbit IgG (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1h to saturate any open antigen-binding sites on the anti-rabbit biotinylated IgG, followed by unconjugated monovalent Fab fragment of goat anti-rabbit (final concentration 0.025mg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) to prevent binding from future secondary IgG. Next, sections for CGRP labeling were incubated with rabbit anti-CGRP serum overnight in a humid atmosphere. On the 3rd day, the sections for PGP 9.5 labeling were incubated with goat anti-guinea pig Alexa 488 fluorescent antibody conjugate (1:500; Invitrogen, Carlsbad, CA) for 1h, while those for CGRP were with goat anti-rabbit Alexa 647 (1:500; Invitrogen, Carlsbad, CA) for 1h. All antisera used were diluted in PBS containing 1% normal goat serum and 0.5% Triton X-100. Before, between, and after each incubation step, the sections were washed five times for 5min in PBS. Finally, all sections were dehydrated through an alcohol gradient (70%, 80%, 90%, and 100%), mounted with a 2:1 preparation of benzyl-alcohol-benzyl-benzoate (BABB), pH=7, and viewed with an MRC-1024 Confocal Imaging system (Bio-Rad, Richmond, CA, USA).
3. Results
3.1. Behavior experiments
3.1.1. Mechanical testing of the paw
In ASIC3 +/+ mice, a significant increase in the number of withdrawals to an 0.4mN force occurs 24h after injection of 3% carrageenan into the knee joint and lasts 2 weeks both ipsilaterally (F1,29=30.1, p=0.0001) and contralaterally (F1,29=11.1, p=0.002) (Fig. 1A). There was also a difference between the ASIC3 +/+ and the ASIC3 −/− mice both ipsilaterally (F1,29=69.5, p=0.0001) and contralaterally (F1,29=54.9, p=0.0001). In ASIC3 −/− mice there was a significant (p<0.05) reduction in the number of responses to mechanical stimuli at all time periods after induction of inflammation for both the ipsilateral and the contralateral hindpaws. There were no differences for sex of the animal in their responses to mechanical stimuli before and after the induction of inflammation.
Fig. 1:
(A) The mechanical sensitivity of the paw is shown before and after joint inflammation in ASIC3 +/+ and ASIC3 −/− mice for the ipsilateral (circles) and the contralateral (squares) hindpaws. The number of responses to an 0.4 mN von Frey filament were significantly less in the ASIC3 −/− mice after inflammation when compared to the withdrawal thresholds from ASIC3 +/+ mice for both the ipsilateral and the contralateral hindpaws. ∗ P < 0.05. (B) The mechanical withdrawal threshold of the knee joint before and after joint inflammation in ASIC3 +/+ and ASIC3 −/− mice for the ipsilateral (circles) and the contralateral (squares) knees. There was no difference between ASIC3 +/+ and ASIC3 −/− mice with decreases in withdrawal thresholds occurring 24 h after joint inflammation in both groups.
3.1.2. Thermal testing of the paw
In both ASIC3 +/+ and ASIC3 −/− mice, there was a significant difference across time for the changes in the withdrawal thresholds to radiant heat ipsilaterally (F1,29=7.9, p=0.009) but no difference between ASIC3 +/+ and ASIC3 −/− mice. The paw withdrawal latency to heat decreased ipsilaterally 24h after induction of inflammation and remained decreased for two weeks after induction of knee-joint inflammation (data not shown). In ASIC3 +/+ mice, baseline withdrawal thresholds averaged 8.24s±0.58 and decreased to a maximum of 6.93s±0.64 72h after induction of inflammation. There were no differences between male and female mice, and no differences on the contralateral limb.
3.1.3. Mechanical testing of the knee joint
In both ASIC3 +/+ and ASIC3 −/− mice, there was a significant difference across time for the changes in the compression-withdrawal threshold of the knee joint ipsilaterally (F1,14=9.2, p=0.009) but no difference between ASIC3 +/+ and ASIC3 −/− mice (Fig. 1B). The time course for the decrease in withdrawal thresholds of the knee joint was different from the other measures. There were maximal decreases 24h after induction of inflammation and were significantly decreased from baseline at both 24h (ASIC3 +/+ p=0.004; ASIC3 −/− p=0.0001) and 72h (ASIC3 +/+ p=0.005; ASIC3 −/− p=0.002) for ASIC3 +/+ and ASIC3 −/− mice. By 1 week the compression-withdrawal thresholds were similar to baseline numbers and were fully resolved by 2 weeks. The decrease in compression-withdrawal threshold of the knee joint was also only found for the ipsilateral side of the ASIC3 +/+ or ASIC3 −/− mice and did not extend to the contralateral hindlimb.
3.2. Histology
The synovium of the knee joint of the uninflamed joint (Fig. 2A and B) was examined for ASIC3 innervation (Fig. 2C) by examining co-localization with the neural marker PGP 9.5. Surprisingly, there was no co-localization of ASIC3 with PGP 9.5 in the synovium of the knee joint in animals without inflammation. There was, however, ASIC3 localization to the synoviocytes (Fig. 2D) in ASIC3 +/+ mice that did not occur in ASIC3 −/− mice (Fig. 2E). Similarly, there was no co-localization of ASIC3 with either PGP 9.5 or CGRP in the synovium of the knee joint in whole specimens of the knee joint in animals without inflammation. (Figs. 3 and 4). In contrast, ASIC3 was located in afferent fibers innervating the knee joint 24h after induction of joint inflammation. In some fibers, ASIC3 co-localized with either PGP 9.5 or with CGRP afferent fibers in the knee joint (Figs. 3 and 4).
4. Discussion
The current study shows that mechanical hyperalgesia of the paw after induction of knee joint inflammation does not develop in ASIC3 −/− mice when compared to ASIC3 +/+ mice. These data agree with prior studies that show mechanical hyperalgesia of the paw after muscle insult, by acid or inflammation, does not develop in ASIC3 −/− mice [22,23]. Further, the current study also shows that there was no difference between ASIC3 +/+ mice and ASIC3 −/− mice in their responses to heat stimuli similar to that observed after muscle inflammation [23]. These data therefore suggest that secondary mechanical hyperalgesia, i.e. increased response to noxious stimuli outside the site of injury, depends on activation of ASIC3 in primary afferent fibers.
The current study showed no differences between ASIC3 −/− and ASIC3 +/+ mice in the mechanical hyperalgesia of the inflamed knee joint, i.e. primary hyperalgesia. Previous studies show that inflammatory hyperalgesia of the paw, both mechanical and heat, is also similar between ASIC3 −/− and ASIC3 +/+ mice [1,7,13]. Thus, although we previously interpreted these data as a difference between cutaneous and muscle insult [22,23], the current data support the conclusion that primary hyperalgesia is unaffected by loss of ASIC3. Further, the data together suggest that secondary hyperalgesia develops in response to peripheral activation of ASIC3. In support, central sensitization does not develop in ASIC3 −/− mice [22]. Specifically, receptive fields of dorsal horn neurons do not expand after muscle insult; and the responses of dorsal horn neurons to mechanical stimuli are reduced in ASIC3 −/− mice when compared to ASIC3 +/+ after muscle insult [22]. Further re-expression of ASIC3 into primary afferent fibers innervating muscle of ASIC3 −/− mice restores the development of secondary hyperalgesia [23]. Together these data suggest that activation of ASIC3 at peripheral sites sends nociceptive information to the central nervous system to result in sensitization of dorsal horn neurons, and the consequent secondary hyperalgesia. It further suggests that ASIC3 at peripheral sites does not result in direct nociceptor sensitization that contributes to primary hyperalgesia.
The current data also show that there was no ASIC3 in primary afferent fibers innervating the synovium in animals without joint inflammation. This was surprising but supports the notion that low levels of ASIC3 are found in peripheral tissues when compared to brain. Previously, Gitterman et al. [4] showed that muscle expresses 16% of the amount of ASIC3 mRNA in comparison with expression levels of the brain. However, approximately 50% of DRG express ASIC3 and there is clear localization of ASIC3 in primary afferents innervating skin and muscle in animals without tissue injury. It is possible that under normal conditions, ASIC3 serves a functional role in cutaneous and muscle receptors as a mechanosensor and/or a pH sensing ion channel. There are clear differences in responses of cutaneous rapidly adapting mechanosensors and in Aδ nociceptors in ASIC3 −/− mice when compared to ASIC3 +/+ mice [13]. Specifically, Aδ mechanonociceptors show decreased responsiveness and increased threshold to mechanical stimuli [13] in ASIC3 −/− mice. Further, 80% of ASIC3-positive DRG neurons innervating muscle also express CGRP, and nerve terminals co-expressing ASIC3 and CGRP are found in primary afferent fibers innervating muscle arterioles in animals without tissue injury [8]. ASIC3 co-localization in muscle arterioles is found mostly in large diameter afferent fibers, and not in thin diameter nociceptors [8]. In contrast, few proprioceptors express ASIC3 [8]. It is yet unknown if primary afferent fibers innervating deep tissue afferents, i.e. muscle and joint, show similar changes in mechanosensors and nociceptors in ASIC3 −/− mice electrophysiologically; or if expression patterns are similar in muscle and joint. Our data suggest that before inflammation ASIC3 is not expressed at detectable levels in primary afferent fibers innervating the knee joint. After inflammation, however, there is expression of ASIC3, and this expression partially co-localizes with CGRP.
This increased expression of ASIC3 after inflammation suggests there is an upregulation of ASIC3 in DRG in response to tissue inflammation. In support, prior studies show ASIC3 mRNA is upregulated in DRG after complete Freund’s adjuvant inflammation of the paw or after placing nucleus pulposis on the nerve root [10,12,26], suggesting there may be an increase in ASIC3 protein after inflammation. Application of inflammatory mediators to DRG neurons (nerve growth factor, serotonin, interleukin-1, arachidonic acid, and bradykinin), mimics this increased expression of ASIC3 mRNA in DRG after inflammation [6,25]. Further, application of these inflammatory mediators to DRG neurons in culture results in an increase in the number of neurons expressing ASIC currents, including ASIC3-like currents, and increase co-expression of ASIC3 and TRPV1 [6]. For the ASIC3-like currents there is an increase in current density and an increase in the number of neurons expressing ASIC3 [6]. These changes in ASIC3 current should increase acid-induced excitation of ASIC3 containing neurons and increase the number of neurons capable of responding to acid.
Surprisingly, in animals without inflammation, there was detectable ASIC3 in synoviocytes of the knee joint. This role of ASIC3 in synoviocytes is unclear and at present speculative. However, other ion channels, i.e. TRPV1, thought to be neuronal in nature, are also found in synoviocytes [3,5,27]. Application of capsaicin to human synoviocytes increases intracellular calcium and application of the TRPV1 receptor antagonist, capsazepine, blocks the increase in intracellular calcium [5]. The role of ion channels in synoviocytes may be to regulate expression of the extracellular matrix substances such as hyaluronan. Indeed hyaluronan is released from synoviocytes in response to stretch [9] and the inflammatory cytokine interleukin-1 decreases synthesis of glycosaminoglycans from synoviocytes [15]. Thus, inflammation and activation of ion channels could modulate the extracellular matrix to maintain adequate lubrication. There may also be direct influences on the levels of ASIC3 by cell–cell contact between synoviocytes and the DRG neurons. Recently, von Banchet et al. [27] showed an increased expression of neurokinin-1, bradykinin-2 receptors, and TRPV1 in DRG in co-cultures of synoviocytes and DRG when compared to DRG alone. In this study, the increased NK1 expression in co-cultures was mimicked by application of the supernatant from synoviocytes. However, the increased expression of bradykinin-2 receptors and TRPV1 was not mimicked by application of the supernatant from synoviocytes [27]. Taken together, these studies suggest that synoviocytes not only secrete substances that interact with DRG but that there also may be cell–cell interactions between synoviocytes and DRG. ASIC3 expression might similarly be affected by secretions from the synoviocytes into the extracellular matrix and/or by cell–cell interactions between synoviocytes and sensory neurons.
In summary, ASIC3 plays a role in the development of secondary, but not primary, hyperalgesia. ASIC3 −/− mice show differences in the development of secondary, but not primary, mechanical hyperalgesia as compared to ASIC3 +/+ mice. Further ASIC3 is located in primary afferent fibers after joint inflammation, but not before joint inflammation. Thus, joint inflammation should result in release of inflammatory mediators that would sensitize primary afferent fibers, increasing expression of ASIC3 in primary afferent fibers innervating synovium. The decreased pH that occurs after inflammation would activate ASIC3 on primary afferent fibers innervating joint, increasing the input to the spinal cord resulting in central sensitization manifested behaviorally as secondary hyperalgesia of the paw.
Acknowledgements
Supported by the National Institutes of Health AR053509.
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