Background
Anecdotal evidence of a link between weather changes and arthritis symptoms has abounded for centuries; several studies have shown that the vast majority of arthritic patients believe their condition to be weather-sensitive [
1,
2]. In rheumatoid arthritis, low temperatures in particular are thought to be associated with exacerbated pain [
2‐
4], though this association is contested [
5]. One particular issue in trying to delineate this phenomenon is that it is often difficult to distinguish the effect of cold independently from other weather variables, such as pressure and humidity, or indeed the psychological effect of inclement weather [
5].
To date, there have been few studies to assess the effect of cold on pain in arthritis. Rats with complete Freund’s adjuvant (CFA)-induced arthritis have an exaggerated hyperalgesic response and sensitized primary afferents after exposure to cold [
6,
7], though the underlying mechanisms are not fully understood. One potential mediator is the transient receptor potential ankyrin 1 (TRPA1) channel, shown to be essential for acute cold hypersensitivity in hindpaw inflammatory models [
8‐
10]. TRPA1 is a ligand-gated non-selective Ca
2+ transducer expressed on neuronal and non-neuronal cells [
11,
12]. The channel is activated by temperatures ranging from 10 °C to 17 °C [
12,
13], though its role as a cold sensor in vivo is controversial [
14]. Conversely, TRPA1 has well-established roles in inflammatory pain, and can be activated by a wide range of endogenous reactive compounds generated by oxidative stress, including hydrogen peroxide [
15]. We have previously demonstrated that TRPA1 mediates tumour necrosis factor alpha (TNF-α)-induced inflammatory pain by modulating mechanical hyperalgesia via both the central and peripheral nervous systems [
16]. In addition, TRPA1 plays an important role in noxious mechanosensation in normal, inflamed and osteoarthritic models [
17], and we have observed a sustained mechanical hyperalgesia in wild-type (WT) but not TRPA1 knockout (KO) mice with CFA-induced mono-arthritis [
16].
As well as sensing noxious stimuli, leading to the perception of pain, another key function of sensory nerves is their ability to release potent vasoactive neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), into the surrounding tissue, leading to neurogenic inflammation [
18]. We have shown that TRPA1 activation leads to neuropeptide-dependent vasodilatation [
19]. Arthritic patients exhibit alterations in the function of their microvasculature [
20], and decreased blood flow in the synovial joint has been observed in rat models of CFA-induced arthritis [
21‐
23]. While the link between environmental cold and blood flow in arthritic joints is not clear at present, rheumatoid arthritis has been associated with Raynaud’s syndrome, which is defined by episodic ischemia of the extremities in response to cold, and defective blood flow regulation [
24].
Here, we have established a model of 1 h exposure to 10 °C environmental cold, and used it to investigate the changes in pain sensitivity and blood flow in the knee joint of CFA-induced mono-arthritic mice. Using both TRPA1 antagonists and KO mice, we have investigated whether TRPA1 is linked to these changes. Our results reveal that TRPA1 influences vascular responsiveness and pain sensitivity following cold exposure.
Discussion
It is not known by what mechanism environmental cold affects arthritis sufferers, though it is often reported as an exacerbating factor for their condition. In this study, we have used the CFA-induced mono-arthritis model with exposure to cold (10 °C for 1 h), to investigate the effect of acute environmental cold exposure on symptoms. We show for the first time that after this acute exposure, bilateral pain sensitivity occurs in the knee joints of cold-exposed arthritic mice 2 weeks after induction of arthritis, and that this is dependent on the cold sensor, TRPA1. Importantly, we also reveal that increased blood flow is seen in the CFA-treated knee joint after cold exposure. This is also dependent on TRPA1, and involves the vasodilatory neuropeptides, SP and CGRP.
We used a range of behavioural pain measurements in our model. Secondary mechanical hyperalgesia was observed after induction of CFA-induced mono-arthritis, and was similar in both RT- and cold-exposed animals, in keeping with previously published reports [
16,
34]. Bilateral thermal hyperalgesia was detected 2 weeks after unilateral CFA injection, irrespective of temperature. Contralateral effects during arthritis are commonly seen both in animal models and humans [
35,
42,
43] so this was not a surprising result. Interestingly, using the pressure application measurement technique to record primary mechanical hyperalgesia of the knee joint, we have shown that mice maintained at RT had a significant decrease in threshold only in their contralateral saline-treated joint and not in the ipsilateral CFA-treated joint 2 weeks after arthritis induction. Mechanisms underlying ipsilateral and contralateral pain differ [
27,
44], with the latter thought to arise from central neurogenic mechanisms [
42,
45]. After induction of unilateral arthritis, spontaneous antidromic action potentials have been recorded in contralateral sensory nerves [
42,
45] and contralateral inflammatory cytokine generation has been observed [
27]. One week after arthritis induction, mice maintained at RT exhibited pain sensitivity in the CFA-treated joint but not in the saline-treated joint (Fig.
2b). Different time courses between ipsilateral and contralateral pain have commonly been reported [
16,
34,
35], with resolution of ipsilateral pain occurring as the pain on the contralateral side starts to manifest. We have previously shown in a model of TNF-α-induced mechanical hypersensitivity that peripheral and central TRPA1 channels are important at different time points during the development of ipsilateral and contralateral pain hypersensitivity [
16]. Importantly, the cold-exposed animals exhibited hyperalgesia in both joints at 2 weeks, indicative of cold-induced pain exacerbation.
We have previously demonstrated a role for TRPA1 in the secondary mechanical hyperalgesia associated with CFA-induced mono-arthritis [
16], but until now no study has assessed a role for TRPA1 in arthritic pain exacerbation caused by environmental cold. Though there is still discussion over whether and how TRPA1 can act as a cold sensor for nociceptive responses in vivo [
14], there is much evidence linking TRPA1 to cold hypersensitivity arising during inflammatory or neuropathic conditions [
8‐
10,
46]. We provide evidence that the bilateral pain in arthritic animals exposed to cold is TRPA1-dependent, as no significant hyperalgesia, when measured with the pressure application measurement device, was observed in both contralateral and ipsilateral knee joints of mice treated with HC-030031 (Fig.
2c). Surprisingly, TRPA1 WT mice did not show any threshold differences between saline- and CFA-treated joints. The TRPA1 genetically modified mice were generated on a mixed genetic background containing both C57BL/6 J and B6129PF2/J strains. Different mouse strains are known to have differing nociceptive sensitivities [
16,
47] and several groups have reported issues with detecting pain differences specifically in C57BL/6 mice [
48,
49]. The variability in pain thresholds was greater in TRPA1 WT mice than KO mice 2 weeks after arthritis induction, and may be indicative of increased stress from the handling and restraint necessary for pressure application measurement. Despite the problems with the TRPA1 WT mice, data using the TRPA1 antagonist suggests TRPA1 does play a role in the bilateral pain observed after cold exposure. It is possible that TRPA1 is not directly activated by cold in this study. Our studies in skin suggest that TRPA1 very quickly senses noxious cold [
30], however, here we have a longer time course which may facilitate lipid metabolism. It is well established that cold exposure activates the sympathetic nervous system resulting in liberation of free fatty acids (FFA) and glycerol, from triglyceride hydrolysis in adipose tissue, which are then released in the circulation and mobilized for energy [
50]. However, we are not aware of possible interactions between FFA and/or glycerol with TRPA1, although, we have previously shown an interaction with TRPA1 and the sympathetic nervous system’s principal neurotransmitter, noradrenaline [
30]. Therefore, it is likely that the results observed in this study are upstream of lipid metabolism.
Vascular remodelling in the joint is a common feature of arthritis, with the growth of blood vessels from the subchondral bone into articular cartilage [
51]. Sensory nerve growth occurs together with the angiogenesis, linking vascular effects to the development of joint pain [
39,
51,
52]. We used two different techniques to examine blood flow in the knee joints of mono-arthritic animals maintained at RT or exposed to cold. FLPI allows real-time imaging of blood flow specifically in the synovial membrane of the knee joint, whereas clearance of i.art. injection of
99mTechnetium is a measure of total blood flow in the entire joint. Both techniques showed a reduction in blood flow in CFA-treated joints compared to contralateral saline-treated joints in animals maintained at RT, in keeping with previous reports [
21‐
23]. This is also consistent with clinical observations of low microcirculatory flow in arthritic joints of patients with rheumatoid arthritis, and is thought to be due to increased microvascular resistance, as a result of inflammation-induced dysfunction [
20].
The primary objective of this study was to investigate the effects of environmental cold on arthritis and its associated mechanisms. In contrast to animals maintained at RT, the CFA-treated joint of cold-exposed animals did not exhibit reduced blood flow compared to the saline-treated joint as shown using the FLPI and
99mTechnetium clearance techniques. Indeed, using FLPI, we recorded increased blood flow in the CFA-treated joint compared to saline-treated joint in cold-exposed CD1 mice. FLPI specifically detects changes in blood flow in the synovial membrane by illuminating the region with a laser and recording speckle patterns produced by moving red blood cells, thus generating flux values correlating to real-time blood flow [
29]. Therefore, this is a more sensitive method of quantifying blood flow than
99mTechnetium clearance, which measures the rate of clearance of
99mTechnetium from the intra-articular cavity into the blood stream, and can be affected by various factors [
53]. Indeed, no changes in clearance rates were observed after pretreatment with HC-030031 in cold-exposed animals, however with FLPI, pretreatment with HC-030031 prevented the increase in blood flow seen after cold exposure in vehicle-treated animals, suggesting the involvement of TRPA1. In addition, TRPA1 KO mice have significantly reduced blood flow in their CFA-treated joints compared to their WT counterparts.
As yet, the functional importance of these changes in blood flow after cold is not clear, so it is not known whether these blood flow changes would be detrimental or beneficial to the arthritic joint. However, as well as altering blood flow, we have shown that TRPA1 blockade reduces joint pain, and this would be a positive effect.
We have previously shown that activation of TRPA1 causes vasodilatation that is neuropeptide-dependent [
19]. Thus, we examined a role for SP and CGRP in the cold-induced increase in blood flow. Systemic administration of both the SP NK
1 receptor antagonist, SR140333, and the CGRP antagonist, CGRP
8–37, prevented the increase in blood flow observed in CFA-treated joints after cold exposure. However, the antagonists also decreased blood flow in the contralateral saline-treated joints, suggesting that SP and CGRP are involved in general blood flow changes after cold, independently of inflammation.
We examined mRNA expression levels of TRPA1, TAC-1 and CGRP from joint tissues in mice maintained at RT or exposed to cold, 2 weeks after arthritis induction, in order to detect the start of any potential changes caused by acute cold exposure. A recent study has shown that expression of the growth factor, vascular endothelial growth factor (VEGF), and the inflammatory mediator, interleukin-1 (IL-1), are increased in cartilage cells from rats with CFA-induced arthritis after exposure to low temperatures [
54]. TRPA1, TAC-1 and CGRP are all highly expressed in DRGs, and we observed no significant differences in their expression under any of the treatment conditions. The only gene altered by CFA treatment in RT-exposed animals was TAC-1, which was significantly reduced in the synovial membrane of the ipsilateral joint. This is perhaps surprising, given that SP-positive nerve fibres are present in the synovium, and increased sensory neuronal growth is known to occur in arthritic joints [
39]. However, other groups have reported a reduction in SP-containing synovial nerves during inflammatory arthritis, suggesting that these nerves may be destroyed by proteolytic enzymes released from inflammatory cells during the course of the disease [
55]. TRPA1 has been suggested to be expressed in non-neuronal cells, including vascular cells [
11] and synoviocytes [
56], although lack of a selective anti-murine TRPA1 antibody has limited studies of protein expression. We found low levels of TRPA1 mRNA expression in the patellar cartilage and synovial membrane in all samples, supporting the concept that TRPA1 gene expression is present in the joint. Whether this expression arises from neuronal or non-neuronal sources, is unclear at this stage. Cold exposure increases the expression of TRPA1 and CGRP mRNA in the patellar cartilage of the CFA-treated joint but decreased levels of TRPA1 mRNA are observed after cold in the synovial membrane from the CFA-treated joint. Vascular remodeling leads to destruction of the synovial lining, and is associated with neuronal growth into the cartilage in arthritic joints [
51]. However, it would be surprising if structural changes could account for differences between RT-exposed control joints and joints exposed to cold for just 1 hour. Although we are presently unable to account for the differences in gene expression observed in this study, it is possible that these changes have a direct impact on blood flow and pain sensitivity under cold exposure. Indeed, it has been suggested that cold exposure leads to increased expression of inflammatory mediators in arthritic joints, such as VEGF, a key angiogenic factor, [
54]. Hence expression of key inflammatory and vascular factors may represent the initial step towards altered joint pathology, leading to an exacerbation of arthritic symptoms following prolonged exposure to cold. Interestingly, a recent study detected increased levels of TRPA1 expression in the skin of subjects with higher pain thresholds, and this expression was regulated through differential methylation of the TRPA1 promoter [
57]. Thus, TRPA1 may contribute to pain sensitivity through tight regulation of its expression.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EF, FR, CS, LL, RS, JB, AA, and KA carried out the blood flow and behavioural studies. EF, SJS, and MA carried out the RT-PCR. CG and SB helped with the knockout mouse studies. EF, FR, and SDB participated in the design of the study, performed the statistical analysis and drafted the manuscript. SDB, MM, and JK conceived of the study, and participated in its design and coordination. All authors revised and approved the final manuscript.