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. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Arthritis Rheumatol. 2014 Sep;66(9):2380–2390. doi: 10.1002/art.38724

TRPA1 contributes to chronic pain in aged mice with CFA-induced arthritis

Sheldon R Garrison 1, Cheryl L Stucky 1,*
PMCID: PMC4149259  NIHMSID: NIHMS617491  PMID: 24891324

Abstract

Objective

Investigate age-related differences in mechanical sensitivity and determine the contribution of transient receptor potential ankyrin 1 (TRPA1) to mechanical hypersensitivity during chronic inflammation in young and aged animals.

Methods

Mechanical sensitivity in young (3-month) and aged (24-month) wild-type (TRPA1+/+) and TRPA1-deficient (TRPA1-/-) mice was measured behaviorally for 8-weeks following injection of Complete Freund's Adjuvant (CFA) into the plantar hindpaw. Histological analysis and hindpaw measurements evaluated inflammation. Ex-vivo skin-saphenous nerve preparations quantified C-fiber sensitivity.

Results

In naïve wild-type mice, aged animals were less sensitive to mechanical stimuli than young. Afferent recordings from TRPA1-/- mice indicate that TRPA1 contributes to the normal mechanical sensitivity in both age groups. Following CFA injection, both young and aged TRPA1+/+ mice exhibited mechanical hypersensitivity. Development of mechanical hypersensitivity was delayed until week 4 in young TRPA1-/- mice, when they exhibited a sharp decrease (9-fold) in mechanical thresholds. In contrast, CFA-injected aged TRPA1-/- mice did not exhibit mechanical hypersensitivity at any time during the entire 8-weeks. Recordings of C-fibers supported these findings and showed that action potential firing increased in both young (25%) and aged (60%) TRPA1+/+ mice 8 weeks after CFA. Interestingly, mechanical firing increased markedly in C-fibers from young TRPA1-/- mice (80%) but not in C-fibers from aged TRPA1-/- mice after CFA.

Conclusions

These data reveal marked differences in long-term mechanical behavioral sensitivity of aged and young mice, and suggest that TRPA1 may be a key contributor to the transition from acute to chronic inflammatory mechanical pain and nociceptor sensitization selectively in aged mice.

Introduction

In humans, the aging process results in progressive degeneration of body tissues that is orchestrated by a complex interaction between the environment and self. These changes are most outwardly visible in the skin, which is constantly exposed to harsh environmental factors that increase the normal breakdown of tissue integrity. In rodents, intrinsic, age-dependent morphological changes that occur at the cellular level include decreased cell proliferation (1), decreased collagen I and elastin protein synthesis by fibroblasts (2), reduced innervation (3) and loss of Meissner's corpuscles (4). Additionally, there are progressive structural changes in rats that begin by 15 months, and include a moderate (10-15%) decline in lumbar DRG neurons (5). By 33 months, there is substantial loss of both unmyelinated and myelinated peripheral fibers (6). These architectural changes may attenuate somatosensation by limiting the range of sensitivity to tensile forces in the skin, decreasing the peripheral input to the spinal cord, and thereby contributing to a decline in tactile and thermal sensitivity (7, 8).

The declining somatosensory perception with advancing age may also be a result of changes at the molecular level. Sensory neurons exhibit decreased conduction velocity (9, 10) and changes in expression levels of ion channels that participate in signal transduction, signal amplification and action potential propagation. Specifically, the sodium channel subtype 1.8 (NaV1.8), which participates in action potential depolarization in nociceptors, and the TRP Vanilloid 1 (TRPV1) channel, the principal noxious heat detector, are decreased at the protein level in the somata and sensory terminals of aged mice (8). These molecular changes support the findings that general somatosensation declines in humans with advanced age (7, 11).

However, decreased tactile and thermal sensitivity with age is somewhat counterintuitive to the documented increase in frequency and severity of pain with age (12). Many older patients have chronic inflammatory conditions such as rheumatoid arthritis, osteoarthritis, gout and lower back pain that are associated with chronic pain. Despite the prevalence of chronic pain with age, mechanisms underlying the pain in aged populations have been little investigated at the molecular, cellular, systems or behavioral levels.

Mechanical hypersensitivity to touch, movement or pressure is the most common attribute of stimulus-evoked pain with inflammatory conditions. The identity of bona fide somatosensory mechanotransduction channels and proteins is not yet confirmed (13). However, one channel, TRPA1, has generated considerable interest for its role in mediating the mechanical hypersensitivity associated with tissue inflammation. In experimental models of acute (1 to 3 days) inflammation using the pro-inflammatory agent Complete Freund's Adjuvant (CFA), mechanical responsiveness is enhanced at both the primary afferent terminal and behavioral levels (14-16). This heightened mechanical sensitivity is accompanied by an increase in TRPA1 mRNA expression levels in dorsal root ganglion (DRG) neurons (17), and TRPA1 antagonists inhibit both the enhanced afferent firing and the behavioral hypersensitivity (14, 16). CFA can also be used as a long-term “chronic” (≥ 3 weeks duration) inflammation that can lead to adjuvant-, or CFA-induced arthritis, which shares many pathological features of human rheumatoid arthritis (18, 19). In this model, mechanical hypersensitivity occurs in wild-type, but not TRPA1-deficient mice (20), suggesting that TRPA1 contributes to the development and maintenance of arthritic pain.

To the best of our knowledge, there have been no reports of the contribution of TRPA1 to any type of acute or chronic (non-resolving) pain in aged animals. In humans, chronic pain is often classified as pain that lasts for at least 3 months (21), and can last for years in many affected individuals. As most animal studies are completed within 1 week of the induction of inflammation, there is a dearth of research on animal models of pain that would be consistent with true chronic inflammatory pain in humans. Understanding the role of TRPA1 during chronic inflammation and arthritic states in an aged population is particularly important given the continuously increasing mean age of the Western world population, the high prevalence of inflammatory pain conditions that this demographic suffers, and their growing need for costly health care services. Therefore, we sought to understand the functional contribution of TRPA1 to mechanical sensitivity in both naïve (non-injured) and chronically-inflamed states, in young (3-month) and aged (24-month) wild-type and TRPA1-deficient mice.

Materials and Methods

Animals

Adult mice were used, which were either wild-type (TRPA1+/+) or global null mutants (TRPA1-/-) on a C57BL/6J genetic background. Mice were divided into young (3-6 month; average 3 months) and aged (≥ 18 month; average 24 months) groups. Mouse genotype was confirmed by PCR of tail DNA. Mice were anesthetized with isoflurane and euthanized by cervical dislocation. Animals were maintained and experimental protocols approved by the Medical College of Wisconsin, and performed in accordance with the Institutional Animal Care and Use Committee. Experimenters were blinded to mouse genotype throughout the histological and behavioral experiments.

CFA-induced paw inflammation

To induce long-term inflammation leading to CFA-induced arthritis, a single 5 μL injection of 1 mg/mL CFA (Sigma Aldrich, St. Louis, MO) was injected subcutaneously into the plantar hindpaw. A single intradermal injection of CFA in this location over an extended period of time (2 weeks) has been demonstrated to induce a rheumatoid arthritis-like pathology in the hindpaw of C57BL/6 mice (22). Controls were injected in the same location with 5 μL phosphate buffered saline (PBS). Paw edema was measured using digital calipers. We scored paws at baseline and on each behavioral testing day using a slightly modified version of what has been previously described (20, 23). Specifically, arthritis development and progression was assessed using a system ranging from 0 to 3: 0, normal; 1, mild swelling and redness in a small area; 2, moderate swelling and redness across the paw; 3, severe swelling, redness, rigidity (stiffness) and deformity of the paw. Paw stiffness and rigidity were measured qualitatively by hand. Mice were euthanized at week 8. Coronal paraffin sections were stained with hematoxylin and eosin (H&E). CFA-induced arthritis was confirmed histologically based on leukocyte infiltration and bone deformation. Tissue sections were scored using qualitative observation of leukocyte infiltration and bone deformation in a system ranging from 0 to 3: 0, no inflammation; 1, mild leukocyte infiltration near the injection site; 2, moderate leukocyte infiltration throughout the paw with observable bone deformation; 3, dense leukocyte infiltration throughout paw with observable bone deformation (20). The clinical and histological scores, 0-6, were combined for a total arthritic score. Because histology was performed at 8 weeks post-CFA-injection, we could not conclusively determine when CFA-induced arthritis first became fully developed. However, other rodent studies using a single CFA injection to the hindpaw, or the hindpaw and tail root together, have demonstrated that features consistent with arthritis begin by 2 weeks (22, 24-26).

Behavior

Mechanical paw withdrawal thresholds of the ipsilateral CFA-injected paw were determined by the Up-Down method (27). Paw withdrawal frequency was used to determine bilateral mechanical sensitivity, where a suprathreshold 3.31 mN von Frey filament was applied 10 times to the plantar surface of each hindpaw. Data was collected at baseline (BL), on post-injection day two (2d), and weeks 1 (1w), 2 (2w), 4 (4w), 6 (6w) and 8 (8w). Baselines were collected from young TRPA1+/+ (n = 10) and TRPA1-/- (n = 10) mice. The same mice were tested at each time point throughout 8 weeks. To avoid learned behavioral changes, we waited at least 3 weeks to continue. We divided this group into randomized sub-groups and increased the n's of CFA- and PBS-injected groups to 10 each by adding young naïve mice. Due to limited numbers of aged mice, we first collected baselines from TRPA1+/+ (n = 10) and TRPA1-/- (n = 11) aged mice and then divided each group into CFA- and PBS-injected subgroups. The CFA-injected subgroup contained TRPA1+/+ (n = 6) and TRPA1-/- (n = 5) aged mice. The PBS-injected subgroup contained TRPA1+/+ (n = 4) and TRPA1-/- (n = 4) aged mice. Three CFA-injected mice died during the study; one CFA-injected young TRPA1+/+ mouse (day 2), one aged TRPA1+/+ mouse (week 2) and one aged TRPA1-/- mouse (week 4) died. Behavioral data from these mice before death were included in the study

Teased fiber skin-nerve recordings

Recordings were performed 8 weeks after CFA or PBS injection. The ex-vivo hairy skin- saphenous nerve preparation was utilized to determine mechanical response properties of single, identified cutaneous primary afferent fibers in TRPA1+/+ and TRPA1-/- mice as described (28, 29). Briefly, preparations were dissected and placed corium side up in a recording chamber superfused with oxygenated synthetic interstitial fluid at 32 ± 0.5°C. The saphenous nerve was desheathed and teased apart until single fibers could be functionally distinguished. Units with conduction velocities <1.2 m/s were classified as C-fibers and were characterized by mechanical threshold.

Quantification of mechanical response properties in afferent fibers

Fibers were recorded for a 2-min period to monitor spontaneous activity. Next, a feedback-controlled, computer-driven mechanical stimulator was used to apply sustained, increasing forces, (5, 10, 20, 40, 100, 150 and 200 mN) for 10 seconds each to the most mechanically-sensitive area of the receptive field. Action potentials were recorded, discriminated and analyzed using LabChart 6 (ADInstruments, Colorado Springs, CO).

Data Analysis

Data was compared between age, treatment and genotypes. The number of mechanically-evoked action potentials across the force range was compared using two-way ANOVA with Bonferroni post-hoc, mechanical thresholds were compared with Mann-Whitney U test, and conduction velocity was compared using student's t test. For behavioral tests, mechanical thresholds and paw withdrawal frequencies were analyzed using Mann-Whitney U, Kruskal-Wallace with Dunn's post-hoc, or a two-way ANOVA with Bonferroni post-hoc as appropriate.

Results

Aged adult mice exhibit decreased mechanosensitivity

Age-related changes in peripheral somatosensation are of importance to tactile acuity, proprioception and balance, and pain detection in the elderly. Because tactile sensitivity has not been well investigated in aged populations, we first quantified mechanical sensitivity in two distinct age groups (3 and 24 months) of naïve (non-injured) mice. Aged mice were less sensitive to mechanical stimuli, with higher paw withdrawal thresholds (5.9 ± 1.1 mN) than young mice (3.1 ± 0.4 mN) (Figure 1A). There were no observable deficits in motor behavior in the aged group, although quantitative locomotor assays were not performed.

Figure 1.

Figure 1

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Young and aged mice respond differently to mechanical stimuli during inflammation. (A) Naïve aged wild-type mice were less sensitive than naïve young wild-type mice. Paw withdrawal thresholds in aged wild-type mice (n = 11) averaged 5.9 ± 1.1 mN compared to 3.1 ± 0.4 mN in young mice (n = 10). (B) Following CFA injection, both age groups exhibited mechanical hypersensitivity in the ipsilateral paw. Beginning by day 2 (2d), young mice exhibited a 4-fold decrease in paw withdrawal threshold while aged mice showed a 4.5-fold decrease. The paw withdrawal thresholds of both age groups remained low through 8 weeks (8w) post-injection. Response to repeated application of a 3.31 mN suprathreshold stimulus was used to measure bilateral sensitivity of the contralateral and ipsilateral paws. (C) Young mice exhibited bilateral hypersensitivity by 4 weeks post-CFA injection. (D) Aged mice exhibited delayed bilateral hypersensitivity with a small increase in response 8 weeks post-CFA injection. Data reported as mean ± s.e.m; n = 8-10 in young mice, n = 4-11 in aged mice. Mann Whitney U test and (B-D) two-way ANOVA with Bonferroni's post-hoc analysis, *p <0.05, **p <0.01 and ***p <0.001.

Aged and young mice exhibit similar mechanical hypersensitivity in a CFA-induced arthritis model

We next asked how young and aged mice respond during the developmental and established phases of this CFA-induced arthritis model. CFA was injected subcutaneously in the plantar hindpaw. This method has been shown to produce arthritic features in the injected hindpaw by 3 weeks post-injection (22). We observed persistent mechanical hypersensitivity in CFA-induced arthritic mice compared to vehicle controls (Figure 1B). Marked mechanical hypersensitivity was evident by day 2 when CFA-injected young mice exhibited a 4-fold decrease in paw withdrawal thresholds (3.1 ± 0.4 mN to 0.8 ± 0.1 mN) and aged mice displayed a 4.5-fold decrease (5.9 ± 1.1 mN to 1.3 ± 0.6 mN). Von Frey thresholds remained low in both age groups throughout the 8-week test period (Figure 1B).

Mice exhibit morphological evidence of arthritic joints

Arthritic development and progression were assessed by scoring injected hindpaws on each day of behavioral testing. CFA-injected mice exhibited persistent paw edema and increased stiffness over the 8-week testing period. Paw scores supported the long-term development of arthritis in CFA-injected mice (Figure 2B). Histological analysis of CFA-injected hindpaws revealed significant leukocyte infiltration and bone deformation (Figure 2A). There were no differences between genotypes or age groups in paw scores (Figure 2C). To measure edema, paw area (width × height) was compared between CFA- and PBS-injected mice at 8-weeks post-injection. Paw area did not differ between baseline and PBS-injected mice at week 8 (data not shown). As expected, the paw area was larger in young CFA-injected wild-type mice (15.5 ± 1.3.1 mm2) than those injected with PBS (9.6 ± 0.3 mm2) (Figure 2D, left). Similarly, paw area was greater in aged CFA-injected wild-type mice (19.9 ± 3.1 mm2) versus those injected with PBS (10.6 ± 0.8 mm2) (Figure 2D, right). Of note, there were no differences in paw area between genotypes within the CFA- and PBS-injected groups (Figure 2D). This suggested that TRPA1 was not required for the development of chronic tissue edema or arthritic-like inflammatory progression.

Figure 2.

Figure 2

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The CFA-induced arthritic inflammatory phenotype in the paw was similar between genotypes and ages. Representative H&E hairy skin cross-sections from wild-type mice at 8-weeks (4x magnification). (A) Aged CFA-injected paw (top left). Aged PBS-injected paw (top right). Young CFA-injected paw (bottom left). Young PBS-injected paw (bottom right). Black arrows indicated increase leukocyte infiltration and purple arrows indicate bone deformation in CFA-injected animals. (B) Qualitative paw scores indicate no differences between age groups or genotypes until week 8. (C) Combined histological and qualitative measurements indicated no differences between genotypes in either age group. (D) The average paw area increased in young CFA-injected TRPA1+/+ (***; n = 12) and TRPA1-/- (***; n = 13) mice when compared to young PBS-injected TRPA1+/+ (n = 9) and TRPA1-/- (n = 8) controls, respectively. No differences between genotypes were observed within CFA and PBS treatment groups (left). The average paw area was similar between CFA-injected aged TRPA1+/+ (n = 6) and TRPA1-/- (n = 3) mice when compared to aged PBS-injected TRPA1+/+ (n = 3) and TRPA1-/- (n = 4) controls, respectively. No differences between genotypes were observed within CFA and PBS treatment groups (right). Data reported as mean ± s.e.m.; **** p <0.05, *** p <0.001.

Aged mice exhibit less bilateral hypersensitivity than young mice

Central sensitization in spinal cord and higher central nervous system (CNS) pathways is a key component of chronic inflammatory pain (30, 31). One behavioral measure that suggests sensitization of central pain pathways is the development of bilateral hypersensitivity following a unilateral trauma or tissue injury (32, 33). To measure bilateral mechanical sensitivity, we repeatedly applied a suprathreshold, punctate 3.31 mN stimulus alternatively 10 times to each hindpaw (34, 35). Both young and aged mice exhibited similar responses between the ipsilateral and contralateral paws at baseline (Figures 1C,D). Young CFA-injected mice exhibited bilateral hypersensitivity by week 4, when their contralateral paw responses increased to 60% from 28% at baseline (Figure 1C). In contrast, bilateral hypersensitivity was not observed in aged animals until week 8, when their contralateral paw responses reached 56% compared to 17% at baseline (Figure 1D). It is possible that the underlying CNS changes involved in bilateral hypersensitivity are more robust in young mice than aged mice. The delayed contralateral paw hypersensitivity in chronically-inflamed aged mice may suggest that their hypersensitivity relies more on continuous peripheral input.

TRPA1 mediates short-term but not chronic pain behavior in young animals

We next asked what mechanisms contribute to the mechanical hypersensitivity in animals with CFA-induced arthritis. A leading candidate is TRPA1, which is expressed in nociceptors, sensitized during inflammation and involved in amplifying mechanical responses (14, 36, 37). Its expression levels are upregulated in dorsal root ganglia (DRG) neurons during inflammation, and pre-treatment with the TRPA1 antagonist HC-030031 prevents the development of mechanical and cold hypersensitivity following inflammation (16, 38).

To determine whether TRPA1 played a role in CFA-induced arthritic pain behavior, we first used naïve animals to confirm baseline differences in mechanosensation between young TRPA1-/- mice and TRPA1+/+ controls. In agreement with previous reports (39, 40) naïve, young TRPA1-/- mice were much less sensitive to mechanical stimuli, exhibiting a higher paw withdrawal threshold (11.9 ± 1.5 mN) than young TRPA1+/+ mice (3.1 ± 0.4 mN) (Figure 3A). More interesting was the way TRPA1-/- mice responded to mechanical stimulation during the development of CFA-induced arthritis. Unlike wild-type mice that exhibited ipsilateral hypersensitivity by 2 days after CFA injection, ipsilateral paw withdrawal thresholds in young CFA-injected TRPA1-/- mice did not differ from PBS-injected controls until week 4, when they exhibited a marked decrease in paw withdrawal threshold (1.3 ± 0.2 mN) compared to baseline (13.5 ± 2.2 mN). Their mechanical thresholds remained low through week 8 (Figure 3B). The sharp drop in threshold in young mice between 2 and 4 weeks was not due to a loss of mice within the experimental group since the same mice were tested throughout the post-injection behavioral time points and none died between 2 and 8 weeks.

Figure 3.

Figure 3

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TRPA1 is important for short-term (≤ 2 weeks) but not long-term (≥ 4 weeks) mechanical hypersensitivity post-CFA injection in young mice. (A) Paw withdrawal thresholds show that naïve TRPA1-/- mice were less sensitive to mechanical stimuli than naïve TRPA1+/+ mice. (B) Young CFA-injected TRPA1+/+ mice exhibited a 4-fold decrease in ipsilateral paw withdrawal threshold on day 2 from baseline (BL) and remained low through week 8. Young CFA-injected TRPA1-/- mice did not differ from baseline until week 4 when there was a sharp 9-fold decrease in threshold. Measurements were recorded from the paw ipsilateral to CFA injection. (C) Bilateral paw withdrawal frequency in CFA-injected TRPA1-/- mice shows responses to 10 applications of a 3.31 mN filament to each paw. Young CFA-injected TRPA1-/- mice did not show contralateral hypersensitivity until week 4. Data reported as mean ± s.e.m.; n = 8-10. (A) Mann Whitney U test and (B, C) two-way ANOVA with Bonferroni's post-hoc analysis, *p <0.05, **p <0.01, *** p <0.001.

We then determined if TRPA1 contributed to the bilateral hypersensitivity in young mice during chronic inflammation. Similar to the delayed sensitization of the ipsilateral, CFA-injected, paw at week 4, the contralateral paw of CFA-injected young TRPA1-/- mice also exhibited increased mechanical response, from 17% at baseline to 36% at 4 weeks (Figure 3C). To determine if the behavioral phenotypes correlated with the severity of gross arthritis inflammation, we evaluated CFA-induced arthritis pathology and found no differences between genotypes at 8 weeks (Figure 2C). As expected, paw area was greater in CFA-injected young TRPA1-/- mice (15.8 ± 0.8 mm2) than those injected with PBS (9.8 ± 0.4 mm2) (Figure 2D, left). Together, these data suggest that in young mice, TRPA1 is required for the short-term (≤ 2 weeks), but not the long-term (≥ 4 weeks), CFA-induced arthritis-associated mechanical hypersensitivity.

TRPA1 mediates chronic pain behavior in aged animals

We next investigated the role of TRPA1 in aged animals. Naïve, aged TRPA1-/- mice exhibited higher paw withdrawal thresholds (9.6 ± 0.9 mN) than wild-type controls (5.9 ± 1.1 mN) (Figure 4A). This suggests that TRPA1 continues to contribute to the normal mechanical response in naïve, aged mice. Similar to our findings in young mice, aged TRPA1-/- mice failed to show ipsilateral mechanical sensitization compared to wild-type controls during the first 4 weeks post-CFA injection. However, in contrast to young mice, aged TRPA1-/- mice did not become sensitized at any time point throughout the 8-week testing period in the ipsilateral paw (Figure 4B). The absence of sensitization in aged mice was not likely due to animal loss, as just one of the four mice from the CFA-injected, TRPA1-deficient group, died between the week 2 and week 4 time points and the standard error was similar before and after the loss of this animal. Furthermore, unlike young mice, bilateral hypersensitivity in the contralateral hindpaw never developed in aged TRPA1-/- mice (Figure 4C). Thus, in aged mice, TRPA1 appears to be required for the short- and long-term arthritis-associated mechanical hypersensitivity.

Figure 4.

Figure 4

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TRPA1 is important for short- (≤ 2 weeks) and long-term (≥ 4 weeks) mechanical hypersensitivity post-CFA injection in aged mice. (A) Paw withdrawal thresholds show that naïve TRPA1-/- mice were less sensitive to mechanical stimuli than naïve TRPA1+/+ mice. (B) Aged TRPA1+/+ mice exhibited a 6-fold decrease in ipsilateral paw withdrawal threshold on day 2 from baseline and remained low through week 8. Aged TRPA1-/- mice did not differ from baseline throughout the 8-week testing period and thus showed no ipsilateral mechanical hypersensitivity. (C) Bilateral paw withdrawal frequency in aged mice shows responses to 10 applications of a 3.31 mN filament to each paw. Contralateral hypersensitivity was not evident in aged TRPA1-/- mice throughout 8 weeks post-CFA. Data reported as mean ± s.e.m.; n = 4-11. Mann Whitney U test and (B, C) two-way ANOVA with Bonferroni's post-hoc analysis, *p <0.05, **p <0.01, *** p <0.001.

Peripheral input contributes to mechanical hypersensitivity in CFA-induced arthritic mice

We next recorded mechanical responses in cutaneous afferent fibers after completion of behavioral testing to determine whether differences in afferent sensitization contributed to the age-related differences in chronic mechanical behavioral hypersensitivity. We recorded from cutaneous C-fibers within the saphenous nerve of CFA-injected and PBS-injected mice to investigate the contribution of TRPA1 in the afferent terminals and hairy skin, where TRPA1 is preferentially expressed (36, 37, 41). Based on histological evidence, we assumed that by 8 weeks, PBS-injected mice were likely fully healed from the plantar-side (glabrous) injection and likely exhibited similar responses to fibers from naïve animals. We found that C-fibers from aged, PBS-injected TRPA1+/+ mice fired a similar number of action potentials to those from young PBS-injected TRPA1+/+ mice across all force intensities (Figure 5A). To better correlate the afferent firing to the manner in which mechanical stimuli were applied with punctate von Frey filaments during the behavioral assays, we specifically analyzed mechanically-evoked action potentials at the force onset (during the ramp phase and first 2 seconds) at 20, 40 and 150 mN. No age-related differences in the onset of mechanical firing in C-fibers were observed in PBS-injected mice (Figure 5B). There were also no differences in von Frey thresholds (Supplementary Table 1). These data suggest that C-fibers contribute minimally to the age-related decline in behavioral mechanical sensitivity of naïve mice.

Figure 5.

Figure 5

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Mechanically-evoked action potential firing in cutaneous C-fibers was similar in young and aged TRPA1+/+ mice in naïve and in CFA-induced arthritic conditions. Action potentials were recorded from cutaneous C-fibers of the saphenous nerve at 8 weeks post-injection of either PBS or CFA. (A) In C fibers from PBS-injected mice, action potential firing during sustained force (5-200 mN, 10 sec each) was similar in young (n = 12, black square) and aged (n = 7, grey square) TRPA1+/+ mice. (B) Further analysis of these fibers revealed no difference in the total number of action potentials recorded during the mechanical ramp (< 1 second) and first 2 seconds of the stimulus at 20, 40 and 150 mN. (C) In C fibers from CFA injected mice, action potential firing during sustained force (5-200 mN, 10 sec each) was similar in young (n = 9) and aged (n = 7) TRPA1+/+ mice. (D) Further analysis of these fibers revealed no difference in the total number of action potentials recorded during the mechanical ramp and first 2 seconds of the stimulus at 20, 40 and 150 mN. Data reported as mean ± s.e.m.; two-way ANOVA with Bonferroni's post-hoc analysis.

Next, we investigated age-associated changes in the firing of cutaneous C-fibers from CFA-injected mice. We found that C-fibers fired a similar number of mechanically-evoked action potentials in both young and aged CFA-injected mice (Figure 5C). Analysis of firing rates at the force onset also revealed no differences between age groups (Figure 5D). There were no changes in von Frey thresholds (Supplementary Table 1). These data indicate that age does not significantly affect C fiber mechanical firing in naïve or inflamed conditions.

TRPA1 mediates primary afferent terminal sensitization in chronically inflamed aged mice

First, we compared mechanical firing in cutaneous C fibers from TRPA1+/+ and -/- PBS controls. The normal response to mechanical stimuli in C-fibers requires TRPA1 in both young (Figure 6B) and aged (Figure 6D) mice. Next, we compared mechanically-evoked action potential firing in young TRPA1+/+ mice 8 weeks after inflammation (arthritic) to young PBS TRPA1+/+ controls and found that CFA induced a 25% increase in overall firing (Figure 6C, left). Strikingly, the action potential firing rate increased by 3.2-fold in young arthritic TRPA1-/- mice compared to young TRPA1-/- controls (Figure 6C, right). This is consistent with the behavioral phenotype at 8 weeks in young mice showing that non-TRPA1 mechanisms mediate the mechanical hypersensitivity (Figure 3B). Interestingly, the contribution of TRPA1 to mechanical firing was different in the aged arthritic population. The C-fibers from aged, arthritic TRPA1+/+ mice responded with more mechanically-evoked action potentials at 150 mN, compared to controls (Figure 6E, left). Most importantly, in contrast to C-fibers from young animals (Figure 6C, right), C-fibers from aged arthritic TRPA1-/- mice showed no sensitization (Figure 6E, right). This finding parallels the behavioral phenotype at 8 weeks, which showed that no sensitization occurred in TRPA1-/- aged mice (Figure 4B). No differences in conduction velocity or mechanical threshold were observed between the ages, genotypes or treatment groups (Supplementary Table 1).

Figure 6.

Figure 6

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Cutaneous C-fibers from aged, arthritic TRPA1-/- mice do not become sensitized to mechanical stimuli. Mechanically-evoked action potentials were recorded from C-fibers at 8 weeks post-injection of either PBS or CFA. Examples of action potential firing from the C-fibers of young (A) PBS-injected TRPA1+/+ mice (left) and CFA-injected TRPA1+/+ mice (right) at 40 mN and 100 mN forces sustained for 10 sec. (B) At 8-weeks post-PBS injection, action potential firing across all force intensities was 1.6-fold lower in young TRPA1-/- mice compared to young TRPA1+/+ mice. (C) In young TRPA1+/+ mice, action potentials across all force intensities increased by 1.6-fold in CFA-injected mice when compared to controls (left). In young TRPA1-/- mice, action potentials increased by 3.2-fold in CFA-injected mice compared to controls (right). (D) At 8-weeks post-PBS injection, action potential firing across all force intensities was 2.8-fold lower in aged TRPA1-/- mice compared to aged TRPA1+/+ mice. (E) In aged TRPA1+/+ mice, action potentials increased by 1.3-fold in CFA-injected mice compared to controls (left). Interestingly, in aged TRPA1-/- mice, unlike young TRPA1-/- mice, there was no difference in C fiber firing between CFA-injected and PBS controls (right). Data reported as mean ± s.e.m.; two-way ANOVA with Bonferroni's post-hoc analysis, *p <0.05, **p <0.01 and ***p <0.001. For clarity, action potential firing from PBS-injected mice was repeated from Figure 5.

In summary, these data suggest that TRPA1 contributes to short-term mechanical hypersensitivity during the development of CFA-induced arthritis in both young and aged animals. However, by 4 weeks, in young animals, non-TRPA1 mechanisms take over mediating the mechanical hypersensitivity. In contrast, in aged mice, TRPA1 continues to be needed for the chronic mechanical hypersensitivity beyond 4 weeks. Thus, TRPA1 appears to be required for the chronic pain behavior in aged, but not young, arthritic mice.

Discussion

General somatosensory decline in aged populations seems paradoxical to the reported increase in frequency and severity of chronic pain that often occurs in older humans (7, 12, 42). The mechanisms underlying these phenomena are poorly understood and studies using aged animal models in somatosensory systems are severely lacking. It is imperative that studies begin investigating aged animal models because the average age of the US population is steeply increasing and health problems associated with chronic pain occur frequently within this demographic. Therefore, demands on and costs to the health care system will continue to increase. Here we report that TRPA1 is essential for the normal behavioral and afferent mechanical responses in both young and aged mouse populations. More remarkably, we identified an age-dependent role for TRPA1 in arthritic-like pain behavior, in that aged CFA-injected TRPA1-deficient mice had markedly lower mechanical hypersensitivity and no mechanical sensitization of nociceptor afferent terminals compared to young mice.

The extended time-course of our study, from the developing through established phases of CFA-induced arthritis, allowed us to tease apart the contribution of TRPA1 in this painful, chronic inflammatory condition in both young and aged mice. As a result, we identified a time- and age-dependent role for TRPA1 in rheumatoid arthritis-like pain behavior. Previous research has shown that TRPA1 has a role in mechanical hypersensitivity during acute (≤ 2 days) and short-term (≤ 2 weeks) inflammation (14, 16, 20, 38). However, to our knowledge, all of these studies have been limited to young animals. Here, we show that TRPA1 is central to the mechanical hypersensitivity during the first two weeks of inflammation in both young and aged animals, and that week 4 represents a critical turning point in neuroinflammatory pain. Moreover, we showed that aged TRPA1-deficient mice did not develop long-term sensitization through 8 weeks, while young TRPA1-deficient mice developed sensitization by 4 weeks. Interestingly, this timeline is consistent with TRPA1-mediated pancreatitis pain in young mice, where TRPA1 contributes to acute but not chronic (≥ 3 weeks) pain (43). Taken together, this data suggests that TRPA1 may be critical for the transition to chronic pain in aged, but not young, individuals.

The importance of continuous peripheral input to the maintenance of chronic pain has been debated, and there is no clear consensus (44, 45). Thus, determining the relative contribution of central versus peripheral mechanisms is the crux of determining how of TRPA1 mediates chronic inflammatory pain. Studies in both young humans and rodents have consistently demonstrated that hyperalgesic responses can be diminished using peripherally-restricted analgesics that decrease nociceptor sensitization and spontaneous activation during inflammation (14, 46). For instance, during acute inflammation, peripherally-targeted delivery of the lidocaine derivative QX-314 specifically to TRPA1-expressing fibers results in a substantial decrease in sensitivity to mechanical stimuli in mice (14). Therefore, it may be possible to target peripheral afferent fibers and other peripheral tissues where TRPA1 is expressed in order to decrease chronic arthritic pain. More importantly, our study suggests that target-based drug discovery for age-related pathologies, such as rheumatoid arthritis, should incorporate aged subjects into the experimental design.

The central nervous system may also contribute significantly to nocifensive behavior during chronic inflammation, and may underlie the marked differences between young and aged mice. Our study did not directly assess central sensitization mechanisms. However, we did find that the development of bilateral hypersensitivity, which is used as one indicator of central sensitization (32), was substantially impaired in aged, arthritic TRPA1-deficient animals. In contrast, the onset of bilateral hypersensitivity in young TRPA1-deficient mice was normal when compared to young wild-type controls. Therefore, persistent, sensitized peripheral input may be important for spinal cord sensitization and bilateral hypersensitivity in aged populations. This sensitization may be partially mediated via TRPA1 at both the distal terminals, where mechanically stimuli are detected, and at the proximal terminals where spinal cord signaling occurs. In the spinal cord, TRPA1 has been shown to mediate increased excitatory synaptic transmission of glutamate and substance P from central afferent terminals onto spinal neurons, thereby, increasing synaptic plasticity in the spinal cord of young mice (47, 48). Collectively, our data suggest that there are age-dependent differences in peripheral TRPA1 function, which may shape the communication patterns between the central primary afferent terminals and spinal dorsal horn neurons during chronic inflammation to drive mechanical hypersensitivity.

Together with a substantial body of evidence that TRPA1 mediates inflammatory mechanical pain, our data suggest that TRPA1 may be a particularly effective therapeutic target for inflammatory pain in aged populations. However, more studies on pain disorders in aged animal populations need to be performed in order to better define the role of TRPA1 in chronically painful disorders that affect aged humans. The data presented here suggest that TRPA1 may play a role in mediating the transition from acute to chronic pain in aged animals, and possibly that aged animals may rely more extensively on continued peripheral input to the CNS. Therefore, peripherally-restricted, TRPA1-targeted antagonists may offer therapeutic potential in aged populations with chronic inflammatory pain.

Acknowledgments

We thank Andy Weyer and Katherine Zappia for their careful review and critique of this manuscript. This work was completed with support from the National Institutes of Health grants NS070711 (C.L.S.), NS040538 (C.L.S.) and PA-08-190 Supplemental Award to NS07011 (S.R.G.).

Footnotes

The authors affirm that there has been no financial support or other benefits from commercial sources for the work reported in the manuscript, or any other financial interests that any of the authors may have, which could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work.

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