To examine the effect of BIB4096 pretreatment on the CSD-evoked meningeal afferent mechanosensitization, we analyzed data collected from 21 afferents (11 A-delta and 10 C); all of which displayed consistent responses at baseline. In BIBN4096-treated animals, CSD-evoked mechanical sensitization in 11/21 afferents (5/11 A-delta and 6/10 C, see Fig. 8A, B). This rate of response was not different from that observed in animals treated with vehicle (7/10 A-delta and 9/13 C; P = 0.35 overall; P = 0.39 for the A-delta afferent population; P = 0.69 for the C afferent population, Fisher exact test). The magnitudes of the TH and STH sensitization responses in the BIBN-treated group were not different from those observed in the control group (TH, Δ1.6 ± 0.5 spikes/s; range 0.3–4.8 spikes/s vs Δ1.8 ± 0.4 spikes/s; range 0.3–4.8 spikes/s, P = 0.74 Mann Whitney test; STH, 1.6 ± 0.1 fold; range 1.2- to 2.3-fold vs 1.6 ± 0.1 fold; range 1.2- to 2.0-fold; P = 0.92; Mann Whitney test). BIBN4096 also did not affect the onset latency for the CSD-evoked sensitization. In the BIB4096-treated group, TH sensitization latency averaged 35.6 ± 6.3 minutes (range 15–60 minutes) and was not statistically different from that observed in the control group (21.0 ± 3.6 minutes, range 15–45 minutes; P = 0.09; Mann–Whitney test; Fig. 8C). The onset latency for the STH sensitization in the BIBN4096 group was 31.5 ± 5.7 minutes (range 15–60), which was also not statistically different from that observed in the control group (24.2 ± 3.2 minutes; range 15–60 minutes; P = 0.25; Mann–Whitney test; Fig. 8D). The duration of the TH sensitization response in the BIBN4096-treated group was 73.8 ± 9.9 minutes (range 30–120 minutes) and was also similar to that observed in the control group (71.3 ± 10.5 minutes; range 30–105 minutes, P = 0.89; Mann–Whitney test; Fig. 8E). Finally, the duration of the STH sensitization response in the BIBN4096 group was 64.5 ± 9.8 minutes (range 30–120 minutes) and was not statistically different from that observed in the control group (73.9 ± 8.7 minutes; range 30–120 minutes; P = 0.54; Mann–Whitney test; Fig. 8F).
The main findings of the study suggest that (1) acute stimulation of meningeal afferents with high K+ concentration promotes a delayed and prolonged increase in their ongoing activity but does not increase their mechanosensitivity, (2) systemic pretreatment with BIBN4096, a CGRP receptor antagonist, blocks the K+-related prolonged activation of meningeal afferents, and (3) a similar BIBN4096 treatment does not inhibit the prolonged activation or mechanical sensitization of meningeal afferents in response to CSD.
Our previous finding that local or systemic application of vasodilating doses of CGRP does not activate or sensitize meningeal afferents38 seems to contradict the current data. The possibility that the levels of CGRP elaborated in response to the K+ stimulation in the present study exceeded the concentrations of CGRP used in our previous work may be entertained. However, previous data suggest that stimulation of meningeal afferents, either electrically or chemically with a mixture of inflammatory mediators or with 50 mM K+ leads to increased CGRP levels at the picomolar range12,20,59—much lower than the micromolar range that failed to influence meningeal afferent responsiveness in our previous study. Differences in the durations of CGRP action between the studies is also an unlikely explanation. In our previous study local CGRP action (measured using changes in local blood flow) lasted for at least 10 minutes when CGRP was applied locally and for ∼15 minutes following systemic application. Thus, the duration of CGRP action in our previous work was likely to be longer than that induced by the release of CGRP in response to the ∼1 minutes excitation of the afferents by the K+ stimulus in the present study. As a potential explanation for the seemingly discrepancy between the 2 studies, we propose that the process underlying the K+-evoked prolonged afferent activation, which requires CGRP, is multifactorial. The release of CGRP from activated meningeal afferents is likely to be accompanied by the release of other neuropeptides, and potentially by other factors such as glutamate. These mediators, in turn, could act upon meningeal immune, vascular, and Schwann cells, which produce the final algesic mediators that enhance the activity of meningeal afferents. We proposed that CGRP action plays a key part in this cascade of events, through a synergistic effect,5,6 by reinforcing or enhancing the release of other algesic mediators from meningeal nonneuronal cells which express CGRP receptors.36
While CSD can be efficiently induced by a cortical pin-prick stimulus, another common induction method, which has been used in numerous in vivo studies of headache mechanisms, involves epidural application of K+ at high concentration (usually 1M). Our finding of acute and prolonged activation of meningeal afferents following epidural K+ stimulation at a much lower dose, that does not promote CSD, thus have important implications for the interpretation of previous studies that used K+ stimulation to study the effect of CSD on trigeminal/meningeal pain (eg, Refs. 2,60,65,73). Future studies that use the CSD paradigm to study related trigeminal responses and their underlying mechanisms thus should be conscientious about the CSD induction method used. The use of less invasive methods that induce CSD remotely, such as optogenetics,31 which may not influence meningeal afferents responsiveness per se may provide a better choice to study mechanisms of CSD-related meningeal nociception and its involvement in migraine headache.
The authors have no conflicts of interest to declare.
Supported by grants from the NIH/NINDS (NS086830, NS078263 to D.L.).
Parts of the manuscript have been presented previously only in an abstract form.
Author Contributions: J. Zhao: performed research and analyzed data. D. Levy designed research, analyzed data and wrote the manuscript.
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