Sustained stimulation of β2- and β3-adrenergic receptors leads to persistent functional pain and neuroinflammation - PubMed (original) (raw)

Sustained stimulation of β2- and β3-adrenergic receptors leads to persistent functional pain and neuroinflammation

Xin Zhang et al. Brain Behav Immun. 2018 Oct.

Abstract

Functional pain syndromes, such as fibromyalgia and temporomandibular disorder, are associated with enhanced catecholamine tone and decreased levels of catechol-O-methyltransferase (COMT; an enzyme that metabolizes catecholamines). Consistent with clinical syndromes, our lab has shown that sustained 14-day delivery of the COMT inhibitor OR486 in rodents results in pain at multiple body sites and pain-related volitional behaviors. The onset of COMT-dependent functional pain is mediated by peripheral β2- and β3-adrenergic receptors (β2- and β3ARs) through the release of the pro-inflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Here, we first sought to investigate the role of β2- and β3ARs and downstream mediators in the maintenance of persistent functional pain. We then aimed to characterize the resulting persistent inflammation in neural tissues (neuroinflammation), characterized by activated glial cells and phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK). Separate groups of rats were implanted with subcutaneous osmotic mini-pumps to deliver OR486 (15 mg/kg/day) or vehicle for 14 days. The β2AR antagonist ICI118551 and β3AR antagonist SR59230A were co-administrated subcutaneously with OR486 or vehicle either on day 0 or day 7. The TNFα inhibitor Etanercept, the p38 inhibitor SB203580, or the ERK inhibitor U0126 were delivered intrathecally following OR486 cessation on day 14. Behavioral responses, pro-inflammatory cytokine levels, glial cell activation, and MAPK phosphorylation were measured over the course of 35 days. Our results demonstrate that systemic delivery of OR486 leads to mechanical hypersensitivity that persists for at least 3 weeks after OR486 cessation. Corresponding increases in spinal TNFα, IL-1β, and IL-6 levels, microglia and astrocyte activation, and neuronal p38 and ERK phosphorylation were observed on days 14-35. Persistent functional pain was alleviated by systemic delivery of ICI118551 and SR59230A beginning on day 0, but not day 7, and by spinal delivery of Etanercept or SB203580 beginning on day 14. These results suggest that peripheral β2- and β3ARs drive persistent COMT-dependent functional pain via increased activation of immune cells and production of pro-inflammatory cytokines, which promote neuroinflammation and nociceptor activation. Thus, therapies that resolve neuroinflammation may prove useful in the management of functional pain syndromes.

Keywords: Allodynia; Astrocyte; Beta 2-adrenergic receptor (β(2)AR); Beta 3-adrenergic receptor (β(3)AR); Beta-adrenergic receptor (βAR); Catechol-O-methyltransferase (COMT); Catecholamines; Chronic pain; Epinephrine; Extracellular signal-regulated kinases (ERK); Functional pain; Hyperalgesia; Idiopathic pain; Microglia; Mitogen-activated protein kinase (MAPK); Neuroinflammation; Norepinephrine; Tumor necrosis factor alpha (TNFα); p38.

Copyright © 2018 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Conflicts of Interest: The authors declare no competing interests.

Figures

Figure 1

Figure 1. Sustained stimulation of β2- and β3ARs results in hypersensitivity to mechanical stimuli that persists for 3 weeks following cessation of OR486

Animals receiving OR486 for 14 days exhibit (A) mechanical allodynia and (B) mechanical hyperalgesia, lasting up to 21 days following the cessation of OR486. The OR486-induced pain behavior is prevented by co-administration of ICI118,551+SR59230A on day 0. Vehicle/Vehicle n=11, OR486/Vehicle n=7; Vehicle/ICI&SR n=8; OR486/ICI&SR n=4 males per group. Data are shown as mean ± SEM. **P < 0.01, *P < 0.05 versus vehicle/vehicle.

Figure 2

Figure 2. Plasma cytokines decrease, while CSF cytokines increase 1-3 weeks following cessation of OR486

Plasma levels of TNFα, IL-1β, and IL-6 decrease over the course of 35 days, while CSF levels of these pro-inflammatory cytokines increase over the course of 35 days (source-by-time interactions are P = 0.0078, 0.021, and 0.0098, respectively). The insets further show the percent change in CSF vs plasma pro-inflammatory cytokine levels on day 35 compared to day 0. Plasma on days 0-35 n=6, CSF on days 0-35 n=6 males per group. Data are shown as mean ± SEM, *P < 0.05, #P < 0.1 versus plasma.

Figure 3

Figure 3. Sustained stimulation of β2- and β3ARs results in increased microglia and astrocyte activation on days 14 and 21

Quantitative analysis of immunofluorescence intensity demonstrates that rats receiving sustained delivery of OR486 for 14 days, exhibit increased activity of (A) CD11b+ microglia and (B) GFAP+ astrocytes in the spinal dorsal horn on days 14 and 21. OR486-induced activation of microglia and astrocytes is blocked by co-administration of ICI-118,551 + SR59230A on day 0. Day 14 groups: Vehicle/Vehicle n=5 (3 males + 2 females); OR486/Vehicle n=6 (3 males + 3 females); OR486/ICI&SR n=6 (3 males + 3 females); Vehicle/ICI&SR n=5 (3 males + 2 females). Day 21 groups: Vehicle/Vehicle n=6 (3 males + 3 females); OR486/Vehicle n=7 (4 males + 3 females); OR486/ICI&SR n=7 (4 males + 3 females); Vehicle/ICI&SR n=6 (3 males + 3 females). Data are expressed as mean ± SEM. **P < 0.01 versus vehicle/vehicle.

Figure 4

Figure 4. Sustained stimulation of β2- and β3ARs results in increased phosphorylation of spinal cord p38 and ERK on days 14 and 21

Quantitative analysis of immunofluorescence intensity demonstrates that rats receiving sustained delivery of OR486 for 14 days, exhibit increased expression of (A) p-p38 and (B) pERK in the spinal dorsal horn on days 14 and 21. OR486-induced phosphorylation of p38 and ERK is blocked by co-administration of ICI-118,551 + SR59230A on day 0. Day 14 groups: Vehicle/Vehicle n=5 (3 males + 2 females); OR486/Vehicle n=6 (3 males + 3 females); OR486/ICI&SR n=6 (3 males + 3 females); Vehicle/ICI&SR n=5 (3 males + 2 females). Day 21 groups: Vehicle/Vehicle n=6 (3 males + 3 females); OR486/Vehicle n=7 (4 males + 3 females); OR486/ICI&SR n=7 (4 males + 3 females); Vehicle/ICI&SR n=6 (3 males + 3 females). Data are expressed as mean ± SEM. **P < 0.01 versus vehicle/vehicle.

Figure 5

Figure 5. The p-p38 and pERK MAPKs are predominantly co-expressed with spinal neurons

Immunohistochemical staining showing the expression of (A) p-p38 (green) and (B) pERK (green) with cell markers (red) for microglia (CD11b), astrocytes (GFAP), and neurons (NeuN). Quantitative analysis reveals that the majority (>80%) of p-p38 or pERK positive cells were co-expressed with NeuN+ neurons. N=6 (3 males + 3 females) per group. Data are expressed as mean ± SEM.

Figure 6

Figure 6. COMT-dependent functional pain is reversed by intrathecal TNFα or p38 inhibitors, but not by systemic β2- and β3AR antagonists

Animals receiving OR486 for 14 days exhibit mechanical allodynia and hyperalgesia over the course of 35 days. Systemic delivery of ICI118,551+SR59230A for 14 days beginning on day 7 does not reverse OR486-induced (A) mechanical allodynia or (B) mechanical hyperalgesia. Vehicle/Vehicle n=8; OR486/Vehicle n=7; OR486/ICI+SR n=6 males per group. Intrathecal administration of the TNFα inhibitor Etanercept (C,D) or the p38 inhibitor SB203580 (E,F) for 14 days beginning on day 14 reverses OR486-induced mechanical pain. Insets represent average pain behavior following inhibitor delivery on days 21-35. N=6 males per group. Data are mean ± SEM. **P < 0.01, *P < 0.05 vs. vehicle/vehicle.

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