Antinociception induced by chronic glucocorticoid treatment is correlated to local modulation of spinal neurotransmitter content

Nociception can be modulated at different levels of the CNS through facilitating (pronociceptive) or inhibiting (antinociceptive) central actions [1‐3]. One of the levels where nociceptive modulation takes place is in laminae I–II of the spinal dorsal horn [4], where nociceptors synapse upon interneurons and projection neurons [5, 6]. The transmission of nociceptive information in the dorsal horn involves several events, neuropeptides and fibres. After peripheral noxious stimulation of unmyelinated nociceptors the release of calcitonin gene-related peptide (CGRP) [7], substance P (SP) [8] and somatostatin (SS) [4, 9] is increased although it remains largely unchanged after innocuous stimulation or stimulation of large myelinated fibres [8, 9]. Spinal nociceptive neurons that are excited by CGRP and SP [10, 11] receive numerous synaptic contacts from primary afferent terminals colocalizing these neurotransmitters, whereas non-nociceptive neurons lack synaptic input from boutons with both peptides [12]. Spinal SS [13] and GABA [14] have an inhibitory effect on nociceptive neurons, being present mainly in fibres belonging to local inhibitory interneurons [15].

Acute stress induces analgesia but the effects of chronic stress in nociception are still controversial, with studies reporting hyperalgesia after prolonged stress [16], while others observed analgesia [17]. Recently, we demonstrated that animals submitted to chronic unpredictable stress display antinociception in the tail-flick test [18]; since the plasmatic levels of corticosteroids were increased throughout the entire experimental period, we implicated these hormones in that phenomenon. Corticosteroids can bind to two types of corticosteroid receptors, mineralocorticoid (MR) and glucocorticoid (GR) receptors. In basal conditions, MR display greater occupancy than GR; thus, conditions resulting in elevation of corticosteroids, e.g. stress, will result mainly in increased activation of GR. Importantly, the spinal cord is a corticoid-responsive tissue [19] and within the spinal cord the greatest density of GR and MR occurs in laminae I–II [20]. Of notice, CGRP and SP (but not SS) coexist with corticosteroid receptors in neurons of dorsal root ganglia [21] and some studies demonstrate that an imbalanced corticosteroid milieu may affect neuropeptide content in the DRG [22, 23]. Importantly, corticosteroids are often used as adjuvant analgesics in the management of several types of pain [24‐26]. Taken together, these findings predict a potential influence of corticosteroids in the modulation of spinal nociceptive transmission.

In the premise that a distinctive activation of MR or GR could be responsible for altered levels of neuropeptides involved in spinal nociceptive transmission and, consequently, for diverse pain-like effects we evaluated the density of CGRP, SP, SS and GABA_B2 innervation in the spinal dorsal horn of animals submitted to prolonged administration of CORT (activating both MR and GR) and DEX (a selective ligand of GR). These data were correlated with pain-like behaviour measured through the tail-flick and hot-plate tests.

The present study demonstrates that prolonged administration of corticosteroids decreases nociception. The antinociceptive effect reflects both a decrease of pronociceptive neuropeptide expression and an increased availability of GABA receptors in laminae I–II of the spinal dorsal horn. After 21 days of treatment the decrease in pain-like behaviour was correlated with a decrease in CGRP and an increase in GABA_B2 receptors in the spinal cord of CORT and DEX treated-animals. Interestingly, the antinociceptive effect in the CORT-group vanished after four weeks of treatment (which was paralleled by a restoration of CGRP and GABA_B2 expression towards control levels) while it remained unchanged in DEX-treated rats (which were correlated with a decrease in spinal content of both CGRP and SP and increased availability of GABA_B2). These findings confirm that corticosteroid receptors play a crucial role in the mediation of pain transmission at the spinal cord level.

Pain perception involves the transmission of nociceptive messages from the periphery to the CNS. This transmission can be modulated by acute [16] and chronic stress [16, 17]. Recently, we showed that chronic unpredictable stress, which results in a prolonged elevation of plasmatic glucocorticoid (GC) levels, decreases pain-like behaviour [18]. Most actions mediated by chronic stress are attributed to hypercortisolemia, as the increased secretion of corticosteroids characterizes the sustained phase of the stress response [27]. Because corticosteroids can bind to two types of receptors we decided to further explore the role of each of these corticosteroid receptors on the nociceptive modulation. It is important to note at this point, that the confounding effect of drug potency has been considered, as the doses of each corticosteroid were adjusted accordingly to their glucocorticoid potency. Thus, in this experimental paradigm CORT treatment differs from DEX administration basically in terms of MR activation: while CORT treatment activates these receptors, DEX does not bind to MR and because it shuts-off the endogenous secretion of corticosteroids, MR remains unoccupied [28].

The results observed after prolonged daily treatment with corticosteroids demonstrate that these steroids promote antinociception. GR are likely to mediate this phenomenon since a similar response was observed in DEX-treated animals. The TF test evaluates a spinally organized reflex [29, 30] mediated by C-fibres innervating the tail [31, 32] and motoneurons innervating the three sets of back muscles that control tail movements [33‐35]. In contrast, the HP test involves a supraspinally integrated response, and thus, represents a more complex behavioural response [36]. Such difference in the neuroanatomical substrates implicated in both tests might explain why there was only a trend towards increased HP latencies after 21 days of CORT and DEX treatments.

It is admissible that the influence of GC upon neuropeptidergic innervation results both from direct and indirect actions. Indirect actions may result from altered availability of GABA_B2 receptors in CORT and DEX treated animals on day 21, as GABA_B receptors are well known players in pain modulation [15, 37]. Moreover Kangrga and collegues [38] described that the antinociceptive effect of GABAergic transmission in the spinal dorsal horn results from presynaptic inhibition of the release of excitatory amino acids and neurotransmitters from the primary afferents [14, 39] which is in accordance with our observations that CGRP, a pronociceptive neuropeptide, is decreased in both CORT- and DEX-treated groups. An alternative indirect action of glucocorticoids might occur through the modulatory actions of arachidonic pathways which down-regulate nerve growth factor (NGF); this, in turn, is known to exert an inhibitory effect in both the accumulation and release of CGRP mRNA in nociceptors [40, 41]. In parallel, the direct modulation of glucocorticoids can be ascribed to the fact that approximately one third of the afferents that are immunoreactive to SP or CGRP, also display immunoreactivity to GR [21]. Thus, it is plausible to assume that GR activation of nuclear responsive elements alters the expression of such transmitters in spinal dorsal horn afferents. This hypothesis is further supported by the fact that it was recently shown that stressors decrease CGRP expression in the frontal cortex, hippocampus, occipital cortex and hypothalamus [42].

Curiously, the dissimilarity in pain-like behaviour observed between CORT and DEX groups after 28 days of treatment, suggests that other mechanisms involving MR activation are implicated in the modulation of pain. In fact, the behavioural differences observed between CORT and DEX treatment at 28 days were paralleled by distinct patterns in CGRP, SP and GABA_B2 expression in the superficial dorsal horn: while in CORT-treated animals the expression of both CGRP and GABA_B2 was restored to control levels, DEX treatment resulted in a decreased expression of SP and CGRP and sustained increase in GABA_B2. The explanations for such discrepancy are more complex, as besides the local effects at the spinal cord level, they might involve alterations at the supraspinal level. Indeed, there is a complex feedback system between the neurotransmitters herein studied and GC involving supraspinal processing that is regulated by MR. There is evidence that the activation of MR is correlated with GABA modulation [43] in lamina II [44] of the spinal cord, namely in interneurones [45], and in other supraspinal pain modulating areas such as the rostroventral lateral medulla (RVM) [46] or the periaductal grey matter (PAG) [47]. This effect of GABAergic transmission appears to selectively inhibit the release of SP, but not of CGRP, [15] which may account at least partly for the differences observed between the groups. Another alternative, but not exclusive, mechanism to explain the differential effect of DEX and CORT upon neuropeptidergic spinal expression derives from the specific modulatory effects of MR upon preprotachykinin (PPT), the precursor of SP expression; in fact, MR activation has been shown to positively regulate (up to 50%) mRNA PPT expression in the nervous tissues [48]. The more persistent changes in pain perception induced by DEX treatment and measured by an increase in both the TF and HP latency at day 28 might therefore result from a decrease in SP fibre innervation in the spinal dorsal horn.

Contrary to CGRP, SP and GABA_B2, no effect of GC was observed on the spinal levels of SS. This differential change observed between these neuropeptides illustrates the selectivity of this process, and is likely to be related with the lack of coexistence of corticosteroid and SS in the spinal cord [21]. Interestingly, different neurotransmitters are associated with different roles in pain modulation [5, 49]. In contrast to CGRP/SP, SS is a tonic inhibitor of peripheral nociceptors [50]. Thus, the data herein reported suggests that the effects of chronic corticosteroid treatment on pain perception are associated with changes in the nociceptive transmitting system (CGRP/SP) but would not involve specific alterations in the spinal intrinsic modulatory system (SS).

In addition to their presence in the spinal dorsal horn, both glucocorticoid- [51] and mineralocorticoid- [52] receptors are present also in neurons of a large number of supraspinal sites along the rostrocaudal extent of the neuraxis in the rat. These include several forebrain and brainstem components of the supraspinal pain control system, including areas like the anterior cingulate cortex [53], amygdala [54], paraventricular hypothalamic nucleus [55], periaqueductal grey matter [56], locus coeruleus [57], rostral ventromedial medulla [58], dorsal reticular nucleus [59] and caudal ventrolateral medulla [60]. Taking into account data obtained in the present study on the effect of corticosteroid manipulation upon spinal neurotransmitter content, future studies should explore alterations induced at supraspinal levels. Accordingly, profound structural, physiological and neurochemical alterations have been observed at different forebrain areas following chronic manipulation of corticosteroids [30, 61‐63].

The present study shows that corticosteroids modulate nociception by altering the expression of neuropeptides involved in nociceptive transmission at the spinal cord level. Moreover, we demonstrate differential modulatory actions of different ligands of corticosteroid receptors, which are of relevance for the pharmacological management of those conditions involving chronic pain, in which corticosteroids are recommended as adjuvant analgesics.