Inflammatory processes are important participants in the pathophysiology of low back pain. Previous studies implicated the MR in mediating the inflammation observed in vessels, heart, and renal cortex of rodent models of diabetes and hypertension. There is increasing evidence to suggest that MR activation increases the risk and severity of inflammation (for review see 6
). Nonetheless, to our knowledge, few studies have examined the role of MR activation in pathological pain at the level of the DRG. In this study, we showed that local inflammation of the DRG with zymosan/IFA leads to enhanced pain behaviors starting as early as POD1, when mechanical hypersensitivity and reduced novelty-induced rearing are also observed. Interestingly, the local inflammation of the DRG is accompanied by the rapid nuclear translocation of MR in DRG neurons on POD1, which is generally considered to indicate MR activation. Pain behaviors persisted for at least two weeks in this study (), and were still evident at 8 weeks in our previous study 9
. We evaluated the effect of adding eplerenone simultaneously with Zymosan/IFA, and found that eplerenone partially reversed these pain behaviors: eplerenone-treated animals returned to the pre-inflammation baseline after one week. Consistent with previous reports, these results suggest that MR activation is involved in the local inflammatory process in the DRG, which triggers pain in this model, hence MR blockade has an analgesic function.
The mechanisms by which selective MR blockers such as eplerenone are anti-nociceptive are not completely understood. Eplerenone was only effective when applied locally to the inflamed DRG; the same amount given systemically did not affect pain behaviors, implicating MR receptors in the DRG. Eplerenone may exert its anti-inflammatory effects indirectly by inhibiting the release of pro-inflammatory cytokines from neurons, glia, or immune cells. The MR is known to promote M1 inflammation characterized by high levels of oxidative metabolites and proinflammatory cytokines as well as tissue damage; in a previous study, microarray experiments on POD3 showed upregulation of 6 out of 10 selected M1 markers 21
in locally inflamed DRG. However, our results also suggest that at least some eplerenone effects may be mediated by direct effects on sensory neurons because eplerenone effects were also observed in cultured neurons, where effects of other cell types should be reduced or diluted. We found marked increases in excitability (decreased rheobase, increased number of action potentials during a suprathreshold current injection) of small sensory neurons that could be observed in neurons removed from the inflamed DRG on POD1 and maintained in primary culture for 8 – 12 hours. These were similar to effects previously reported for cells isolated on POD3 10
. The nuclear translocation of MR in inflamed DRG was also preserved in cells cultured on POD1. The increased excitability could be partially reversed by treating the cells in vitro with eplerenone. These results suggest that the MR located in neurons may play important roles in establishing pain directly through excitability increases as are seen following DRG inflammation. One possible mechanism is the activation of the pro-inflammatory nuclear factor-κB transcription factor, which has been shown to mediate pro-inflammatory effects of MR in some other tissues 22–24
; nuclear factor-κB has excitatory and pro-nociceptive effects in DRG neurons (e.g. see25,26
and refs therein).
The finding that eplerenone reduced GFAP expression in satellite glia (generally used as a marker of activation) in inflamed DRG might be attributed to indirect general anti-inflammatory effects, since GFAP upregulation is observed in inflammatory conditions. Activity-dependent somatic release (especially of adenosine triphosphate) from sensory neurons provides a mechanism for neuron-satellite glia communication 27
that may be enhanced in the inflamed DRG, where excitability and spontaneous activity are increased. Since experiments with activity blockers show that abnormal neuronal activity plays a role in satellite glia activation 28,29
, the observed eplerenone effects on neurons could also play a role in the reduced GFAP expression. Alternatively or in addition, eplerenone could also have had a direct effect on the satellite glia. In the central nervous system, glia have been shown to express the GR 30
Our immunohistochemistry results indicated that the MR was not primarily located in the nucleus in normal DRG, translocating there only early after DRG inflammation. For the classical nuclear actions of the MR receptor, such translocation is generally taken as evidence for activation. The observations in normal DRG may seem to contradict the general view that the MR in most tissues should be chronically activated by basal plasma levels of corticosterone (except in tissues such as kidney where corticosterone is enzymatically degraded) – the affinity of the MR for corticosterone is higher than its affinity for aldosterone, so the (much higher) basal plasma levels of corticosterone should chronically activate the MR. RNA for the enzyme that degrades corticosterone in classical aldosterone-sensitive tissues, 11 -hydroxysteroid dehydrogenase type II, is present in DRG 21
though it is not known if this is neuronal or perhaps associated with vascular cells. In addition, the MR can apparently have forms that are less sensitive to corticosterone. For example, studies of MR in brain 31
as well as other tissues 32
suggest that the MR that is in or closely associated with the plasma membrane has a lower affinity for corticosterone; hence this form of the receptor is not chronically activated. This form may mediate some of the fast, excitatory nongenomic effects of the MR in neurons 33
. Further studies are needed to understand what endogenous agonist causes the MR translocation to the nucleus after DRG inflammation in our model. One possibility is that systemic corticosteroid levels increase due to the pain model (as observed in some other pain models, e. g. 34–36
), enough to activate the MR. However, if this were the case, translocation should have occurred in all DRG, but we found that DRGs several levels above the inflamed DRG did not show nuclear translocation on POD1. Alternatively, locally produced endogenous steroids could activate the receptor, or the local inflammation process could somehow modify the MR receptor or associated proteins so that it has sensitivity to basal corticosterone levels more like that observed in other tissues. Finally, it is worth noting that the apparent subcellular localization of the MR can vary depending on what antibody is used, presumably because ligand or interacting proteins may interfere with binding of a particular antibody, or because a given antibody may only recognize certain conformations of the protein18
. The antibody used in this study was derived from an antigen based on the last (C-terminal) 20 amino acids of the MR sequence, which is in the ligand-binding domain. The unexpected pattern of MR localization we observed in normal DRG neurons was somewhat similar to that described in amygdala neurons using an antibody that, like the one we used, recognizes the ligand-binding domain 37
The above discussion assumes that the MR effects studied in the patch clamp experiments were genomic, nuclear effects. Our studies were designed to examine such effects; aldosterone and eplerenone were applied for at least 4 – 8 hours (pretreatment) but were not present during the recording. In addition, our electrophysiological studies were consistent with these assumptions: no effect of eplerenone was observed in normal cells (which had little nuclear receptor), but aldosterone applied to normal cells had excitatory effects. In hippocampal neurons the rapid nongenomic effects of steroids mediated by plasma membrane MR can be rapidly washed out 33
and hence even if present should not have been observed in our recordings. However, in other systems some of the membrane-MR effects can be prolonged, often tending to reinforce the genomic effects 32
. We cannot completely rule out the possibility that longlasting but non-genomic effects contributed to our findings.
The dose-response curve for aldosterone effects in electrophysiological experiments appeared to be an inverted U-shaped, with excitatory effects at 1 and 10 nM. The declining effects at higher doses could be due to activation of the GR receptor. In some tissues including macrophages and microglia 6
and some brain regions 31
, MR and GR have opposing effects. This may also be true at the level of the spinal cord where neuronal MR is also found. In one study, using intrathecal injection, agonists of GR but antagonists of MR had antinociceptive effects 35
, however, the role of spinal GR in pain is controversial ( e. g. 34,36
and references therein).
Inflammation is a component of many different models of both inflammatory and neuropathic pain, not just low back pain models. This study presents evidence that the MR may play important roles in pain acting at the level of peripheral sensory neurons. It will be of interest to determine the possible effects of MR antagonists in other types of pathological pain. The MR is a potentially novel target for pain therapeutics, particularly since drugs targeting MR are already approved by the United States Food and Drug Administration for other clinical uses.