Cranial dural permeability of inflammatory nociceptive mediators: Potential implications for animal models of migraine

Abstract

Background Application of inflammatory mediators to the cranial dura has been used as a method to activate and sensitize neurons in the meningeal sensory pathway in preclinical behavioral studies of headache mechanisms. However, the relatively high concentrations and volumes used in these studies raise the question of whether the applied agents might pass through the dura to act directly on central neurons, thus bypassing the dural afferent pathway. Methods We used a radiolabeling approach to quantify the meningeal permeability of two of the inflammatory mediators, 5-HT and PGE₂, when applied to the cranial dura as part of an inflammatory mixture used in preclinical headache models. Results Both agents could be detected in samples taken four hours after dural application in the cerebrospinal fluid (CSF) and, in measurements made only for PGE₂, in the central nervous system (CNS) as well. Based on our measurements, we made estimates of the CSF and CNS levels that would be attained with the higher concentrations and volumes of 5HT and PGE₂ that were exogenously applied in previous pre-clinical headache studies. These estimated levels were comparable to or larger than normal endogenous levels, potentially large enough to have physiological effects. Conclusions The finding that the cranial meninges are permeable to the two tested inflammatory mediators PGE₂ and 5-HT raises some uncertainty about whether the behavioral changes observed in prior pre-clinical headache studies with these as well as other agents can be attributed entirely to the activation of dural nociceptors, particularly when the agents are applied at concentrations several orders of magnitude above physiological levels.

Keywords: Inflammatory mediators; headache models; meningeal permeability; rat.

Figure 1

CSF measurements. Measurement of diffusion of tritiated 5-HT ((a), (b)) or PGE₂ ((c), (d)) into the CSF following application of 1 μl of a mixture of inflammatory mediators to a 1 mm diameter area of the cranial dura overlying the frontal cortex (final concentration of the applied solution: 5-HT, BK, HA, all 1 mM, and PGE₂, 100 μM). (a) Tritium measurements, in DPM (mean ± SEM), of 10 μl samples of CSF withdrawn from the cisterna magna prior to (n = 6) and at two and four hours following (n = 6, for each time point) dural application of inflammatory soup spiked with tritiated 5-HT. Two consecutive 10 μl samples were drawn at two and four hours following the dural application. (b) Measurements of the counts, in DPM (mean ± SEM), of 10 μl samples of standard dilutions of the tritated 5-HT (n = 3) for comparison with the measurements of the CSF samples in (a). (c) Same measurement as in (a), in experiments in which the inflammatory mediators were spiked with tritiated PGE₂. A single 35 μl sample was withdrawn four hours following the dural application (n = 9). (d) Measurements of the counts, in DPM (mean ± SEM), of 35 μl samples of standard dilutions of the tritated PGE₂ (n = 4), for comparison with the measurements of the CSF samples in (c). For both 5-Htand PGE₂, the mean of the tritium measurements of the CSF samples was extremely close to that of the 5 × 10⁵ dilution standard (dotted line). Since the tritiated compound was applied to the dura in a 1:1 dilution, we estimate that the diffusion through the dura into the CSF resulted in a further dilution of approximately 2.5 × 10⁵.

Figure 2

CNS measurements. Diagram (a) and plot (b) show the distribution of PGE₂ in the different CNS regions. The “X” in (a) marks the approximate position of the dural application site. Measurements (mean ± SEM shown) were significantly above background for each of the CNS regions (p <0.01), but not for the trigeminal ganglia (NS—not significant).