Tumor necrosis factor-α induces sensitization of meningeal nociceptors mediated via local COX and p38 MAP kinase actions

Abstract

The proinflammatory cytokine TNF-α has been shown to promote activation and sensitization of primary afferent nociceptors. The downstream signaling processes that play a role in promoting this neuronal response remain however controversial. Increased TNF-α plasma levels during migraine attacks suggest that local interaction between this cytokine and intracranial meningeal nociceptors plays a role in promoting the headache. Here, using in vivo single unit recording in the trigeminal ganglia of anesthetized rats, we show that meningeal TNF-α action promotes a delayed mechanical sensitization of meningeal nociceptors. Using immunohistochemistry, we provide evidence for non-neuronal localization of the TNF receptors TNFR1 to dural endothelial vascular cells and TNFR2 to dural resident macrophages as well as to some CGRP-expressing dural nerve fibers. We also demonstrate that meningeal vascular TNFR1 is co-localized with COX-1 while the perivascular TNFR2 is co-expressed with COX-2. We further report here for the first time that TNF-α evoked sensitization of meningeal nociceptors is dependent upon local action of cyclooxygenase (COX). Finally, we show that local application of TNF-α to the meninges evokes activation of the p38 MAP kinase in dural blood vessels that also express TNFR1 and that pharmacological blockade of p38 activation inhibits TNF-α evoked sensitization of meningeal nociceptors. Our study suggests that meningeal action of TNF-α could play an important role in the genesis of intracranial throbbing headaches such as migraine through a mechanism that involves at least part activation of non-neuronal TNFR1 and TNFR2 and downstream activation of meningeal non-neuronal COX and the p38 MAP kinase.

Figure 1

TNF-α evoked sensitization of a C-unit meningeal nociceptor. A. Identification and isolation of a C-unit meningeal nociceptor based on the response latency (11 milliseconds) to electrical stimulation of the dura and a consistent waveform (insert). B. Peri-stimulus time histograms depicting the responses to threshold (S1) and suprathreshold (S2–3) mechanical stimulation of the dura at baseline, before TNF-α application, 30 and 60 min after TNF-α (100 ng/ml), and then 60 min after wash with SIF. The numbers in parenthesis indicate mean firing rate in spikes/sec. The rate of ongoing activity is depicted in all trials prior to S1. Note the persistent sensitization during the wash period.

Figure 2

Time course changes in the average responses of Aδ and C-units meningeal nociceptors following topical application of TNF-α (100 ng/ml) and the effect of co-application of the TNF-α blocker Peg-sTNFR1 (1.5 mg/ml). A. Mean ± SEM response magnitude to mechanical stimulation of the dura using threshold (top) and suprathreshold (middle) pressure stimuli, and ongoing firing rate (bottom) at baseline with SIF, during TNF-α treatment and during the wash with SIF. B. Time course changes in the mean ± SEM responses to TNF-α in the presence of its blocker Peg-sTNFR1. Note the lack of TNF-evoked sensitization in the presence of the blocker.

Figure 3

Effects of topical application of the COX inhibitors SC-560 (100 nM) and NS-398 (10µM) on baseline mechanical responsiveness of meningeal nociceptors and the development of TNF-α evoked mechanical sensitization. A. Median, 10^th, 25^th, 75^th and 90^th percentile changes in threshold (top) and suprathreshold (bottom) responses of meningeal nociceptors to mechanical stimulation of the dura 60 min following application of TNF-α alone (in SIF) and TNF-α in the presence of SC-560 or NS-398. Note the additional inhibition of suprathreshold values following treatment with NS-398. B. Median, 10th, 25^th, 75^th and 90^th percentile changes in baseline threshold (top) and suprathreshold (bottom) responses of meningeal nociceptors following local application of SC-560 or NS-398. Note the inhibition in baseline responsiveness following NS-398 treatment. (* p<0.05 sensitization, # p<0.05, inhibition, Wilcoxon test, 60 min after drug vs. baseline).

Figure 4

TNF-α evoked meningeal nociceptors’ sensitization involves p38 MAP kinase. Median, 10^th, 25^th, 75^th and 90^th percentile changes in threshold (A) and suprathreshold (B) responses of meningeal nociceptors to mechanical stimulation of the dura 60 min following application of TNF-α alone (in the presence of SIF), TNF-α in the presence of the p38 inhibitor SB203580 (SB), or SB203580 alone (in the presence of SIF). Note the blockade of TNF-α evoked sensitization by SB203580 and the lack of effect of this drug on baseline values. (* p<0.05 Wilcoxon test, 60 min after drug vs. baseline). C. Average number of pp38 labeled dural blood vessels observed per visual field following topical application of TNF-α or SIF (# p<0.05, Mann Whitney test TNF-α vs. SIF).

Figure 5

Immunohistochemical localization of TNFR1 in dural blood vessels. Note the localization of TNFR1 with vimentin (vim), COX-1, and pp38 and its lack of co-expression in peripherin immunolabeled dural nerve fibers.

Figure 6

Immunohistochemical localization of TNFR2 in the dura mater. Note the localization of TNFR2 with ED-2 (macrophages) and COX-2 positive cells. Also note the expression of TNFR2 in axonal like structures that are immunolabeled with CGRP.