Neurobiology of Disease

Dissociation between CSD-Evoked Metabolic Perturbationsand Meningeal Afferent Activation and Sensitization:Implications for Mechanisms of Migraine Headache Onset

X Jun Zhao and X Dan LevyDepartment of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, and Harvard MedicalSchool, Boston, Massachusetts 02215

The onset of the headache phase during attacks of migraine with aura, which occur in �30% of migraineurs, is believed to involve corticalspreading depression (CSD) and the ensuing activation and sensitization of primary afferent neurons that innervate the intracranialmeninges, and their related large vessels. The mechanism by which CSD enhances the activity and mechanosensitivity of meningealafferents remains poorly understood, but may involve cortical metabolic perturbations. We used extracellular single-unit recording ofmeningeal afferent activity and monitored changes in cortical blood flow and tissue partial pressure of oxygen (tpO2 ) in anesthetizedmale rats to test whether the prolonged cortical hypoperfusion and reduction in tissue oxygenation that occur in the wake of CSDcontribute to meningeal nociception. Suppression of CSD-evoked cortical hypoperfusion with the cyclooxygenase inhibitor naproxenblocked the reduction in cortical tpO2 , but had no effect on the activation of meningeal afferents. Naproxen, however, distinctly preventedCSD-induced afferent mechanical sensitization. Counteracting the CSD-evoked persistent hypoperfusion and reduced tpO2 by preemp-tively increasing cortical blood flow using the ATP-sensitive potassium [K(ATP)] channel opener levcromakalim did not inhibit thesensitization of meningeal afferents, but prevented their activation. Our data show that the cortical hypoperfusion and reduction in tpO2

that occur in the wake of CSD can be dissociated from the activation and mechanical sensitization of meningeal afferent responses,suggesting that the metabolic changes do not contribute directly to these neuronal nociceptive responses.

Key words: cortical spreading depression; hypoperfusion; hypoxia; migraine; primary afferent; trigeminal

IntroductionMigraine is a multifactorial, episodic neurological disorder and thesecond leading cause of disability worldwide (Vos et al., 2017). It is

now well accepted that the onset of migraine headache requires theactivation and increased mechanosensitivity of trigeminal affer-ent neurons that innervate the intracranial meninges and theirrelated large blood vessels (Strassman et al., 1996; Messlinger,2009; Olesen et al., 2009; Levy, 2012). However, the endogenousprocesses underlying these afferent responses during a migraineattack are incompletely understood.

Migraine with aura, the second-most prevalent migraine sub-type, is thought to involve cortical spreading depression (CSD),

Received Jan. 17, 2018; revised March 15, 2018; accepted April 10, 2018.Author contributions: J.Z. and D.L. designed research; J.Z. and D.L. performed research; J.Z. and D.L. analyzed

data; D.L. wrote the paper.The study was supported by National Institutes of Health Grants NS-086830, NS-078263, and NS-101405 to D.L.

We thank Dr. A. M. Strassman for helpful comments on the manuscript.The authors declare no competing financial interests.Correspondence should be addressed to Dan Levy, Department of Anesthesia, Critical Care and Pain

Medicine, Beth Israel Deaconess Medical Center, DA717; 330 Brookline Avenue, Boston MA 02215. E-mail:dlevy1@bidmc.harvard.edu.

DOI:10.1523/JNEUROSCI.0115-18.2018Copyright © 2018 the authors 0270-6474/18/385053-14$15.00/0

Significance Statement

Cortical spreading depression (CSD)-evoked activation and mechanical sensitization of meningeal afferents is thought to mediatethe headache phase in migraine with aura. We report that blocking the CSD-evoked cortical hypoperfusion and reduced tissuepartial pressure of oxygen by cyclooxygenase inhibition is associated with the inhibition of the afferent sensitization, but not theiractivation. Normalization of these CSD-evoked metabolic perturbations by activating K(ATP) channels is, however, associatedwith the inhibition of afferent activation but not sensitization. These results question the contribution of cortical metabolicperturbations to the triggering mechanism underlying meningeal nociception and the ensuing headache in migraine with aura,further point to distinct mechanisms underlying the activation and sensitization of meningeal afferents in migraine, and highlightthe need to target both processes for an effective migraine therapy.

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an abnormal wave of neuronal and glial activity (Ayata and Lau-ritzen, 2015). CSD has been suggested to mediate the aura phase(Pietrobon and Moskowitz, 2013), and also contributes to theonset of the headache phase, by promoting prolonged activationand increased mechanosensitivity of meningeal afferents (Zhanget al., 2010; Zhao and Levy, 2015, 2016) and the ensuing activa-tion and sensitization of central trigeminal dorsal horn neuronsthat receive their input (Zhang et al., 2011b; Melo-Carrillo et al.,2017). The precise mechanisms by which CSD promotes the ac-tivation and sensitization of meningeal afferents are unclear, al-though dural neurogenic inflammation (Bolay et al., 2002; butsee Zhao and Levy, 2017) and Pannexin-1-related signaling(Karatas et al., 2013) have been hypothesized.

Migraine with aura is associated with cortical hemodynamicchanges, in particular a sustained reduction in cortical blood flow(CBF) contemporaneous with the onset of the headache phase(Olesen et al., 1981, 1982; Lauritzen, 1984; Hadjikhani et al.,2001; Hansen and Schankin, 2017). In animal studies, CSD hasbeen shown to promote a similar prolonged cortical hypoperfusion(Fabricius and Lauritzen, 1993; Fabricius et al., 1995; Piilgaardand Lauritzen, 2009). CSD also leads to transient cortical hypoxiafollowed by prolonged and milder reduction in cortical tissuepartial pressure of oxygen (tpO2) concomitant with the corticalhypoperfusion phase (Takano et al., 2007; Piilgaard and Laurit-zen, 2009).

Reduced tissue oxygenation during acute exposure to highaltitude can promote headache and trigger a migraine attack (Ap-penzeller, 1994; Broessner et al., 2016). Normobaric hypoxia hasalso been shown to trigger migraine in susceptible individuals(Schoonman et al., 2006; Arngrim et al., 2016). These clinicalobservations together with the notion that reduced blood flow andtissue oxygenation can lead to enhanced activity and responsivenessof primary afferent nociceptors that innervate the cornea, viscera,muscle, and skin (Haupt et al., 1983; Mense and Stahnke, 1983;Longhurst et al., 1991; MacIver and Tanelian, 1992; Hillery et al.,2011) prompted us to examine whether the cortical hypoperfu-sion and associated reduction in tpO2 that occur following CSDmight contribute to the prolonged activation and sensitization ofmeningeal afferents. Recent work has shown that the inhibitionof cortical cyclooxygenase abrogates CSD-evoked cortical hypo-perfusion (Gariepy et al., 2017). Here, we investigated whethercyclooxygenase inhibition might also ameliorate the CSD-evokeddecreases in tpO2 and interfere with the associated meningealafferent responses. Having identified such effects, we furthertested whether a preemptive increase in CBF to counteract theCSD-evoked hypoperfusion and reduced tpO2, using the ATP-sensitive potassium [K(ATP)] channel opener levcromakalim(Kleppisch and Nelson, 1995; Shin et al., 2003) might also inhibitthe enhanced responses of meningeal afferents. Our data suggestthat while cyclooxygenase inhibition and K(ATP) channel open-ing can counteract the CSD-related cortical hypoperfusion andreduced tpO2, they exert distinct inhibitory effects on the associ-ated activation and sensitization of meningeal afferents, thusquestioning the direct contribution of these cortical metabolicperturbations to meningeal nociception and the onset of head-ache in migraine with aura.

Materials and MethodsAnimals, anesthesia, and surgical preparation. The current study usedSprague Dawley rats (males; 250 –350 g; Taconic). Rats were housed 3per cage under a 12 h light/dark cycle, in a temperature- and humidity-controlled room in the animal care facility of the Beth Israel DeaconessMedical Center (Boston, MA). Food and water were available ad libitum.

All experimental procedures followed the Guide for Care and Use of Lab-oratory Animal Resources (NIH publication No. 85–23, revised 1996),were approved by the Animal Care and Use Committee of the Beth IsraelDeaconess Medical Center, and were in compliance with the ARRIVE(Animal Research: Reporting of In Vivo Experiments) guidelines. Ani-mals were deeply anesthetized with urethane (1.5 g/kg, i.p.) and mountedon a stereotaxic frame (Kopf Instruments). Core temperature was kept at37.5–38°C using a homoeothermic control system. Animals were intu-bated and breathed spontaneously room air enriched with O2. Physio-logical parameters were collected throughout the experiments usingPhysioSuite (Kent Scientific) and CapStar-100 (CWE). Data used in thisreport were obtained from animals exhibiting physiological levels of ox-ygen saturation (�95%), heart rate (350 – 450 beats/min), and end-tidalCO2 (3.5– 4.5%). A saline-cooled dental drill was used to perform threeseparate craniotomies (Fig. 1A). One craniotomy was used to expose theleft transverse sinus and the posterior part of the superior sagittal sinus, aswell as the adjacent cranial dura, extending �2 mm rostral to the trans-verse sinus. Another small craniotomy (1 � 1 mm) was made in thecalvaria over the right hemisphere, and was centered 2 mm caudal and2 mm lateral to bregma to allow insertion of the recording electrode. Anadditional small burr hole (diameter, 0.5 mm) was drilled to expose asmall area of the frontal cortex to induce CSD (Zhao and Levy, 2015,2016). The exposed dura was bathed with a modified synthetic interstitialfluid containing 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM CaCl2, 10mM glucose, and 10 mM HEPES, pH 7.2. The femoral vein was cannulatedto allow for intravenous administration.

Recording of meningeal afferent activity. Single-unit activity of menin-geal afferents (1 afferent/rat) was recorded from their cell body in theipsilateral (left) trigeminal ganglion using a contralateral approach, asdescribed previously (Zhao and Levy, 2014, 2015, 2016; Fig. 1A) in whicha platinum-coated tungsten microelectrode (impedance, 50 –100 k�;FHC) is advanced through the right hemisphere into the left trigeminalganglion using an angled (22.5°) trajectory. The insertion of the record-ing electrode using this approach does not produce CSD in the left cor-tical hemisphere (Zhao and Levy, 2015, 2016). Meningeal afferents wereidentified by their constant latency response to electrical stimuli appliedto the dura above the ipsilateral transverse sinus (0.5 ms pulse, 5 mA, 0.5Hz). The response latency was used to calculate conduction velocity (CV),based on a conduction distance to the trigeminal ganglion of 12.5 mm(Strassman et al., 1996). Neurons were classified as A-� (1.5 � CV � 5m/s) or C afferents (CV � 1.5 m/s). Neural activity was digitized andsampled at 10 KHz using power 1401/Spike 2 interface (CED). A real-time waveform discriminator (Spike 2 software, CED) was used to createand store a template for the action potential evoked by electrical stimu-lation, which was used to acquire and analyze afferent activity.

Assessment of changes in meningeal afferent mechanosensitivity. Me-chanical receptive fields of meningeal afferents were first identified byprobing the exposed dura with a 2.0 g von Frey filament (Stoelting). Thesite of lowest mechanical threshold was further determined using mono-filaments that exert lower forces (�0.02 g). For quantitative determina-tion of mechanical responsiveness, we applied mechanical stimuli to thesite with the lowest threshold, using a servo force-controlled mechanicalstimulator (Series 300B Dual Mode Servo System, Aurora Scientific),controlled via the power 1401 interface. The stimulus was delivered usinga flat-ended cylindrical plastic probe that was attached to the tip of thestimulator arm. One of three probe diameters (0.5, 0.8, or 1.1 mm) wasselected for each neuron, depending on the sensitivity of the neuron(Levy and Strassman, 2002). Stimulus trials for testing the mechanosen-sitivity of the afferent were made using a custom-written script for Spike2 and consisted of “ramp and hold” stimuli (rise time, 100 ms; stimuluswidth, 2 s; interstimulus interval, 120 s). Each trial included a thresholdstimulus (which normally evoked at baseline 1–3 Hz responses) followedby a suprathreshold stimulus (usually X2 of the threshold pressure). Tominimize response desensitization, stimulus trials were delivered every15 min throughout the experiment (Zhang et al., 2013; Zhao and Levy,2016). Ongoing afferent discharge was recorded continuously betweenthe stimulation trials. Responses to mechanical stimuli were determinedduring at least four consecutive trials before the elicitation of CSD. Datawere analyzed only from afferents that exhibited consistent responses

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(variation of �0.5 Hz for threshold responses and �1.5 Hz for suprath-reshold responses) during baseline recordings (Zhao and Levy, 2016).

Induction of CSD. A single CSD episode was induced by quickly insert-ing a glass micropipette (diameter, 50 �m) �2 mm deep in the cortex for2 s (Zhang et al., 2010). CSD was triggered in the left frontal cortex toavoid potential damage to the meningeal tissue near the tested receptivefield of the afferent under study, which could have led to their activationand/or sensitization (Zhao and Levy, 2015).

Measurement of CSD-evoked changes in CBF using laser Doppler flow-metry. Changes in CBF (Gariepy et al., 2017) were documented using aneedle probe (diameter, 0.8 mm) connected to a laser Doppler flowmeter(Vasamedic). The probe was positioned just above the intact dura, �1mm from the mechanical receptive field of the recorded afferent in anarea devoid of large pial blood vessels (�100 �m). Such a system (usinga near-infrared 785 nm probe and fiber separation of 0.25 mm) is ex-pected to record blood flow changes at a cortical depth of �1 mm (Fab-ricius et al., 1997). The laser Doppler signal was digitized (100 Hz) andrecorded using the power 1401/Spike 2. Laser Doppler recordings wereobtained with the ambient lights turned off. Baseline data were based onrecordings conducted for at least 30 min before CSD.

Measurement of cortical partial pressure of oxygen. Cortical tpO2 wasrecorded using a modified Clark-type polarographic oxygen microelec-trode (OX-10, Unisense) with a guard cathode. The oxygen electrode wasconnected to a high-impedance pico-amperometer (PA2000; Unisense)that sensed the currents of the oxygen electrodes. The tpO2 signal was

digitized (100 Hz) and recorded using thePower 1401/Spike 2 interface. The microelec-trode responded linearly to changes in oxygenconcentration and was calibrated before and af-ter each experiment in both air-saturated salineand in an oxygen-free solution consisting of 0.1 M

of sodium-ascorbate and 0.1 M NaOH dissolvedin saline. This approach has been used previ-ously to record changes in cortical tpO2 in re-sponse to CSD (Takano et al., 2007; Piilgaardand Lauritzen, 2009; Thrane et al., 2013).Changes in tpO2 values were recorded at a cor-tical depth of 100 –200 �m, with the electrodeplaced �0.5 mm away from the laser Dopplerflowmetry (LDF) probe. Baseline tpO2 valueswere recorded for at least 30 min before CSD.

Electrophysiological recording of CSD. A tungstenmicroelectrode (impedance, 1–2�; FHC) wasused to record multiunit activity at a depth of200 – 400 �M in the left visual cortex (V1). Asilver ground electrode was placed in the neckmuscle. Signal was amplified and filtered (20Kx, 300 Hz low frequency; 10 KHz high frequen-cy; DAM-80/BMA400, WPI/CWE), and thendigitized and sampled at 10 KHz. Cortical ac-tivity was recorded simultaneously with CBFchanges, with the recording electrode placed�0.5 mm away from the LDF probe.

Drugs. Naproxen sodium and levcromaka-lim were purchased from Tocris Bioscience.Naproxen sodium was made in 0.9% immedi-ately before administration (10 mg/kg, i.v). Alevcromakalim stock solution (10 mM) wasmade in DMSO and further diluted with syn-thetic interstitial fluid (final concentration, 500�M) immediately before its application. Theconcentration of levcromakalim expected to beattained at the superficial cortex was estimatedto be 100–1000 times lower (Moore et al., 1982;Roth et al., 2014; Zhao et al., 2017).

Data analyses and statistics. All data were an-alyzed and plotted using Prism 7 software andare presented as median (IQR). Group data ingraphs show all data points and are plotted asbox-and-whisker plots with minimum and

maximum values. For CSD-related changes in CBF and tpO2, raw datawere processed with a custom-written script made for Spike 2. Baselinevalues were based on the average of the values collected during the 600 sbefore the induction of CSD. Off-line analyses for afferent responseswere conducted using template matching in Spike 2. Criteria used toconsider meningeal afferent activation and sensitization responses werebased on our previous studies (Zhao and Levy, 2015, 2016). In brief,prolonged increase in the ongoing activity of afferents was considered ifthe firing rate increased above the upper end point of the 95% CI calcu-lated for the baseline mean for �10 min. Acute afferent activation duringthe CSD wave was considered as an increased ongoing discharge rate thatoccurred �30 s following the pinprick and lasted �120 s. Mechanicalsensitization was considered only if the afferent responses changed accordingto the following criteria: threshold and/or suprathreshold responses in-creased to a level greater than the upper end point of the 95% CI calculatedfor the baseline mean; increased responsiveness began during the first 60min post-CSD; and sensitization lasted for at least 30 min. Changes inCSD-evoked responses of meningeal afferents in the presence ofnaproxen or levcromakalim were compared with data obtained in con-trol animals using the same surgical approach and CSD inductionmethod. Control data were obtained from experiments in animalstreated intravenously with the naproxen vehicle (0.9% saline, 60 minbefore CSD induction, n � 24), or locally (i.e., continuous dural bathing)with synthetic interstitial fluid (n � 36). The two datasets were compa-

Figure 1. A, Experimental setup: three skull openings (red ovals) were made. A small burr hole was made over the left frontalcortex to elicit a single CSD event using a pinprick (PP) stimulation. Meningeal afferent activity was recorded in the left trigeminalganglion (TG) using a tungsten microelectrode inserted through a craniotomy made over the contralateral hemisphere. An ipsi-lateral craniotomy was made to expose a part of the left transverse sinus (TS) and superior sagittal sinus (SSS) and their vicinity tosearch for mechanosensitive meningeal afferents. Quantitative mechanical stimuli were delivered to the receptive field of afferentsusing a feedback-controlled mechanical stimulator. A Laser Doppler Flowmeter (LDF) probe was placed over the cortex near thereceptive field of afferents to record changes in CBF and validate the induction of CSD noninvasively. In some animals, we alsorecorded the induction of CSD by monitoring multiunit activity (MUA) of cortical neurons. An oxygen microelectrode was placed inthe superficial cortex to record CSD-evoked changes in cortical tpO2. B, Example of raw data recording depicting the activation of aC afferent meningeal afferent (with an overdrawn spike waveform used for data analysis) following the elicitation of CSD with a PPduring the prolonged reduction in CBF and tpO2. The afferent recording trace represents a peristimulus time histogram (1 sbin-size). Average ongoing activity rate (spikes/s) are denoted in parentheses. The activation phase and associated cortical hypo-perfusion and reduced tpO2 are colored red. LDF values are in arbitrary units (AU). stim, Stimulus.

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rable and therefore combined to increase power. Differences in CSD-evoked afferent activation and sensitization propensities were analyzedusing two-tailed � 2 tests. Within-group and between-group compari-sons were made using the Wilcoxon test and Mann–Whitney U test,respectively. Results were considered to be significant at p � 0.05.

ResultsCSD-evoked prolonged activation of meningeal afferentscoincides with cortical hypoperfusion and reduced tpO2

Protracted cortical hypoperfusion and reduced cortical tpO2

were observed previously (Piilgaard and Lauritzen, 2009; Fords-mann et al., 2013) at time points corresponding to the prolongedactivation of meningeal afferents (Zhang et al., 2010; Zhao andLevy, 2015). To examine whether the CSD-evoked cortical met-abolic perturbations indeed coincide with prolonged meningealafferent activation, we used simultaneous recording of meningealafferent activity with CBF and tpO2 (n � 6). As the example inFigure 1B demonstrates, persistent meningeal afferent activationcould be observed during the prolonged post-CSD cortical hypo-perfusion and reduced tpO2 stage.

Suppression of CSD-evoked cortical hypoperfusion anddecreased tpO2 by naproxen is not associated with reducedactivation of meningeal afferentsWe initially verified that systemic administration of naproxen,which was used to ensure cortical COX (cyclooxygenase) inhibi-tion also under the nerve endings of meningeal afferents brancheslocalized to areas not exposed by the craniotomy, could also blockthe CSD-evoked cortical hypoperfusion, as we previously haveshown using topical administration (Gariepy et al., 2017). Be-cause cortical oxygenation level depends in large part on corticalperfusion (Offenhauser et al., 2005), we further examined whetherblocking the CSD-evoked cortical hypoperfusion with naproxenmight inhibit the reduction in cortical tpO2. When compared withcontrol animals (n � 22), naproxen treatment (n � 22) aug-mented the CSD-related acute cortical hyperemic response andblocked, as expected, the protracted cortical hypoperfusion (Fig.2). Using simultaneous recording of CBF and tpO2, we observedthat the inhibitory effect of naproxen on the CSD-evoked corticalhypoperfusion was associated with altered tpO2 responses toCSD (Fig. 3). In the presence of naproxen (n � 6), CSD evoked aninitial increase in tpO2 [�10.03 mmHg (5.22); W � 6.0; p � 0.03;Fig. 3B, phase 1 and arrowhead in the insert] that was smallerthan in vehicle-control animals [n � 11; �14.18 mmHg (3.87);U � 7.0; p � 0.007; Fig. 3C]. While in control animals the hyper-oxia phase was followed by acute hypoxia [bottom values: 17.66mmHg (18.64); W � 66.0; p � 0.001], which lasted 110.0 s(70.30; Fig. 3A; phase 2), naproxen blunted this hypoxic re-sponse, and instead tpO2 values increased [113.4 mmHg (90.24);W � 21.0; p � 0.015; Fig. 3B, phase 2]. This hyperoxia phasereturned to baseline levels at 15–25 min [59.02 mmHg (39.63);W � 7.0; p � 0.29; phase 3, Fig. 3B]. Overall, when compared withcontrol animals, naproxen inhibited both the acute hypoxia (phase2; U � 0; p � 0.001; Fig. 3D) and prolonged decrease in tpO2 (phase3; U � 3; p � 0.005; Fig. 3E).

We next examined whether the suppressing effects of naproxenon CSD-evoked cortical hypoperfusion and decreased tpO2 mightbe associated with reduced activation of meningeal afferents. A-�afferents recorded from naproxen-treated animals (n � 15) hadbaseline ongoing activity similar to that in control vehicle-treatedanimals [n � 26; 0.04 spikes/s (0.20) vs 0.05 spikes/s (0.25); U �189.0; p � 0.87]. Baseline ongoing activity of C afferents was alsonot different between the naproxen group (n � 16) and control

group [n � 34; 1.08 spikes/s (1.58) vs 0.57 spikes/s (1.35); U �215.0; p � 0.24]. In naproxen-treated animals, CSD evoked acuteactivation in 5 of 15 A-� afferents and 6 of 16 C afferents, ratiosthat were not significantly different from those observed in con-

Figure 2. The effect of systemic naproxen on CSD-induced changes in CBF. A, Changes in CBFfollowing induction of CSD in vehicle- and naproxen-treated animals. B, CBF changes during thehyperemia stage, showing the enhancing effect of naproxen. C, CBF changes during the hypo-perfusion phase, 10 – 60 min following CSD, depicting the inhibitory effect of naproxen.p Values are based on a Mann–Whitney U test. PP, Pinprick.

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trol animals [9 of 26 A-� afferents, 14 of 34 C afferents; � 2 �0.007; p � 0.99 for the A-� afferent population; � 2 � 0.06; p �0.80 for the C afferent populations; � 2 � 0.07; p � 0.79 for thecombined population]. When compared with the control group,naproxen also did not affect the magnitude of the immediateactivation in either A-� afferents [4.2-fold (3.50) vs 3.6-fold(2.10); U � 22.0; p � 0.99] or in C afferents [1.6-fold (0.50) vs2.1-fold (1.40); U � 30.0; p � 0.35, Fig. 4C].

The likelihood of developing a prolonged increase in ongoingafferent activity following CSD in naproxen-treated animals (Fig.5A,B, examples) was also not different when compared with thatobserved in control animals (A-� afferents: 7 of 15 vs 13 of 26,� 2 � 0.04; p � 0.84; C afferents: 7 of 16 vs 22 of 34, � 2 � 1.96; p �0.16; combined population: �2 � 1.427; p � 0.23). Naproxen treat-ment also did not affect any of the CSD-evoked afferent activationparameters: persistent activation developed in A-� afferents witha delay of 25 min (40), which was no different than in controlanimals [10 min (5.0); U � 33.0; p � 0.34, Fig. 5C]. The activa-tion delay observed in C afferents in naproxen-treated animals[15 min (31.25)] was also not different than that in controls [5min (17.5); U � 67.5; p � 0.58, Fig. 5C]. The duration of CSD-evoked prolonged activation was also unaffected by naproxen:A-� afferents were activated for 15 min (15) vs 30 min (22.5) inthe control group (U � 22.0; p � 0.07, Fig. 5D); C-afferents wereactivated for 30 min (31.25) vs 35 min (30.0) in the control group(U � 58.0; p � 0.34; Fig. 5D). Finally, when compared withcontrols, naproxen treatment did not reduce the magnitude of

the afferent activation observed in either population [A-� affer-ents: 2.3-fold (3.0)] vs [3-fold (3.8); U � 39.0, p � 0.64; C affer-ents: 1.8-fold (3.0) vs 1.5-fold (0.9); U � 51.0; p � 0.18, Fig. 5E].In afferents deemed not activated by CSD, the changes in ongoingactivity following CSD were also not different between thenaproxen and control groups, in both the A-� afferent (U � 41.0;p � 0.8) and C afferent (U � 35.0; p � 0.2) populations (Fig. 5F).

Naproxen inhibits CSD-evoked meningealafferent mechanosensitizationWe previously proposed that the activation and mechanosensiti-zation of meningeal afferents that occur following CSD aremediated via distinct mechanisms (Zhao and Levy, 2016). Wetherefore examined next in 8 A-� and 7 C afferents whether theinhibitory effects of naproxen on the cortical hypoperfusion anddecreased tpO2 might be associated with amelioration of thepost-CSD mechanosensitization response. Because the charac-teristics of the CSD-evoked sensitizations of the A-� and C affer-ents are similar (Zhao and Levy, 2016), we combined the datafrom the two afferent populations for comparisons with the dataobtained from vehicle-treated animals (10 A-� afferents and 13 Cafferents). At baseline, the number of dural mechanosensitivereceptive fields and the mechanical responsiveness of afferentsrecorded in naproxen-treated animals were similar to those inthe control group (Table 1). Unlike its ability to inhibit theCSD-evoked activation of meningeal afferents, naproxen blockedthe afferent sensitization responses, at both the threshold level [3

Figure 3. Naproxen inhibits CSD-induced decreases in cortical tpO2. A, B, Time course of changes in tpO2 following CSD induction in control (A) and naproxen-treated animals (B).C–E, Comparisons between CSD-evoked changes in control and naproxen-treated animals during the hyperoxia phase (phase 1), the acute hypoxia phase (phase 2, in control animals), and at 15–25min post-CSD (phase 3). p Values are based on a Mann–Whitney U test. PP, Pinprick.

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of 15 vs 13 of 23; �(2) � 4.97; p � 0.03; Fig. 6C] and the suprath-reshold level [2 of 15 vs 13 of 23; �(2) � 7.09; p � 0.008; Fig. 6].When sensitization was defined as enhanced responsiveness ateither stimulus level, there were also fewer sensitized afferents inthe naproxen group [4 of 15 vs 17 of 23; �(2) � 9.084; p � 0.003].When the responses of all sensitized and nonsensitized units werecombined, naproxen also had an overall inhibitory effect on thechanges in threshold responses [�0 spike/s (0.6) vs �0.39 spikes/s(1.43); U � 90.5; p � 0.01, Fig. 6G] and suprathreshold responses[0.86-fold (0.5) vs 1.2-fold (0.5); U � 22.0; p � 0.004, Fig. 6H].

The K(ATP) channel opener Levcromakalim counteractsCSD-evoked cortical hypoperfusion and reduced tpO2

Naproxen may inhibit the CSD-evoked afferent mechanosensiti-zation indirectly, by blocking the cortical hypoperfusion and re-duced tissue oxygenation. However, it is also possible that theinhibition of sensitization is independent of these cortical meta-bolic effects, since prostanoids (elaborated by CSD), whose syn-thesis is blocked by naproxen, exert a direct sensitizing effect onmeningeal afferents (independent of metabolic effects; Zhang etal., 2007), and naproxen also inhibits afferent sensitization in-duced by a mixture of inflammatory mediators (Levy et al., 2008).We therefore asked next whether opposing the CSD-evoked cor-tical hypoperfusion and the associated decrease in tpO2 using analternative, cyclooxygenase-independent approach might alsoinhibit the afferent sensitization response. We postulated thatlocal application of a potent cortical vasodilator could serve thispurpose and tested the effects of levcromakalim, a selective K(ATP)channel opener with strong cortical vasorelaxation properties

(Reid et al., 1995). In preliminary experiments, we observed thatthe maximum cortical vasodilatory effect of levcromakalim oc-curs within 15–25 min. Therefore, to counteract the CSD-evokedpersistent hypoperfusion, we used a pretreatment approach andstarted levcromakalim treatment 30 min before CSD elicitation.Using this protocol, before CSD induction, levcromakalim (n �28) increased basal CBF [196.10% (72.7); W � 231.0; p � 0.0001,Fig. 7A]. Levcromakalim did not inhibit the elicitation of CSD(n � 3, Fig. 7C), which was associated with a significant corticalhyperemic response, albeit smaller in magnitude when comparedwith the response in control animals [105.3% (1.9) vs 180.3%(61.6); U � 0.0; p � 0.0001; Fig. 7E]. In the presence of levcro-makalim, there was a decrease in CBF following CSD, but valuesdid not attain the hypoperfusion level observed in control ani-mals (Fig. 7F; U � 51.0; p � 0.0001); up to the 45 min post-CSDperiod, values were not different from those observed during thepredrug baseline period [99.56% (39.67); W � 8.0; p � 0.99]and increased afterward [119.1% (54.9)] at 60 min post-CSD(W � 366.0; p � 0.0001).

We next tested whether levcromakalim might also affect theCSD-evoked changes in cortical tpO2 (n � 8). Before CSD elici-tation, levcromakalim increased the basal tpO2 [�43.55 mmHg(35.3); W � 36.0; p � 0.008; Fig. 8A]. In the presence of levcro-makalim, CSD gave rise to a further, brief increase in tpO2 [peak,120.5 mmHg (56.3); W � 36.0; p � 0.008; Fig. 8A, phase 1] witha magnitude similar to that observed in control animals [�18.22mmHg (10.32) vs �14.18 mmHg (3.87); U � 24.0; p � 0.11].While the duration of this hyperoxic phase was shorter than that incontrol animals [70.0 s (30.0) vs 100 s (50.0); U � 11.0; p � 0.05],

Figure 4. Naproxen treatment does not inhibit CSD-evoked acute activation of meningeal afferents. A, B, Examples of experimental trials depicting the acute activation of an A-� afferent (A) anda C afferent (B) following the induction of CSD using a pinprick (PP) stimulus in animals treated with naproxen. Traces represent a post-stimulus time histogram (1 s bin size). The average ongoingactivity rate (spikes/s) is denoted in parentheses. Red areas represent the acute phase of afferent activation during CSD. Raw LDF traces are provided below each recording showing the CSD-evokedincrease in CBF. C, D, Comparisons of the magnitude of change in ongoing activity between afferents recorded in control and naproxen-treated animals depicting activated (C) and nonactivatedafferents (D). p Values are based on a Mann–Whitney U test.

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overall the hyperoxia phase [area under the curve (AUC)] wasnot different between the two groups (U � 33.0; p � 0.39; Fig.8C). Following the brief hyperoxia, tpO2 values decreased tran-siently, reaching hypoxic values [18.5 mmHg (26.36)] that werenot different from those in control animals [17.66 mmHg(18.64); U � 32.0; p � 0.35]. However, when compared with con-trol animals, levcromakalim shortened this hypoxic phase [20 s(27.5) vs 110 s (70.0); U � 3.5; p � 0.0002], yielding a reducedhypoxic response [246.6 mmHg*sec (676.0) vs 2001.0 mmHg*sec(4883.9); U � 7.0; p � 0.002; Fig. 8D]. Levcromakalim also pre-vented the prolonged reduction in tpO2 when compared with con-trol animals (U � 15.0; p � 0.001; Fig. 8B, Phase 3, E), with valuesnot different than baseline at the 5–10 min time point [49.49 mmHg

(43.2); U � 26.0; p � 0.46] and the 10–20 min time point [47.16mmHg (44.51); U � 31.0; p � 0.64].

Levcromakalim inhibits CSD-evoked activation but notsensitization of meningeal afferentsGiven its ability to counteract the cortical metabolic perturba-tions evoked by CSD, we next determined the effect of levcro-makalim treatment on the development of CSD-evoked afferentactivation in 9 A-� and 11 C afferents. In the presence of levcro-makalim, baseline ongoing activity in A-� afferents [0.13 spikes/s(0.31)] and in C afferents [0.28 (0.89)] were not different fromthat in control animals (U � 91.0, p � 0.32 and U � 156.0, p �0.41, respectively). Levcromakalim treatment, however, signifi-cantly inhibited the acute afferent activation [control group:A-� afferents, 0 of 9 vs 9 of 26; �(1) � 4.19, p � 0.04; C afferents,1 of 11 vs 14 of 34; �(2) � 3.85, p � 0.049); combined population:1 of 20 vs 23 of 60; �(2) � 7.937; p � 0.005; Fig. 9C]. Levcro-makalim also inhibited the prolonged afferent activation whencompared with control group [A-� afferents, 1 of 9 vs 13 of 26;�(2) � 4.21; p � 0.04; C afferents, 3 of 11 vs 22 of 34; �(2) �4.717; p � 0.03; combined population: 4 of 20 vs 35 of 60; �(2) �8.82; p � 0.003; Fig. 9D].

Finally, we tested the effect of levcromakalim on the develop-ment of CSD-evoked mechanical sensitization in 7 A-� afferentsand 10 C afferents, which had a similar number of identified duralmechanosensitive receptive fields and baseline mechanical re-sponsiveness as the afferents tested in the control group (Table 1).

Figure 5. Naproxen treatment does not inhibit CSD-evoked prolonged activation of meningeal afferents. A, B, Experimental trials depicting the CSD-evoked prolonged activation of an A-�afferent (A) and a C afferent (B) in animals treated with naproxen. C–E, Naproxen treatment did not affect the onset latency of the afferent activation (C), its duration (D), or its magnitude (E).Changes in ongoing activity rate in nonactivated afferents in naproxen-treated animals were not different than those in controls (F ). p Values are based on a Mann–Whitney U test. PP, Pinprick.

Table 1. Baseline mechanical response properties of meningeal afferents inanimals treated with vehicle, naproxen, or levcromakalim

Receptivefields*

Thresholdresponses**

Suprathresholdresponses***

Control 2 (1) 1.3 (0.7) 7.3 (7.0)Naproxen 2 (1) 0.5 (1.8) 8.0 (6.5)Levcromakalim 2 (1) 1.7 (1.5) 9.0 (4.0)

Data are expressed as the median (IQR). Threshold and suprathreshold neural responses (spikes/s) are based onactivity recorded during a 2 s mechanical stimulus, and represent the average of three to four trials conducted priorto the elicitation of CSD in the presence of the vehicle, naproxen, or levcromakalim. Group differences were analyzedusing Kruskal–Wallis test and were not statistically significant for the three parameters examined. H stands for“Kruskal-Wallis H test”; H(2) � H test with 2 degrees of freedom.

*H(2) � 3.85, p � 0.145; **H(2) � 4.70, p � 0.095; ***H(2) � 3.24, p � 0.198.

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Unlike its effect on the CSD-evoked afferent activation, levcro-makalim treatment did not inhibit the sensitization response(Fig. 10A, example). When compared with control experiments,levcromakalim did not reduce the relative number of afferentsthat were deemed sensitized when using the threshold [10 of 17 vs13 of 23 affected afferents; �(2) � 0.04; p � 0.85] or the suprath-reshold [6 of 17 vs 14 of 23 affected afferents; �(2) � 1.1, p �0.29] stimuli. When sensitization was defined as enhanced re-sponsiveness at either stimulus level, the ratio of sensitized/nonsensitized was also not different when compared with thatobserved in control animals [11 of 17 vs 17 of 23; �(2) � 0.2, p �0.66]. When compared with controls, levcromakalim also did notdecrease the magnitude of the sensitization responses to thresh-old stimuli [�1.73 spikes/s (3.36) vs �1.3 spikes/s (2.18); U �38.5; p � 0.10; Fig. 10D] or suprathreshold stimuli [1.39-fold(0.5) vs 1.37-fold (0.54); U � 34.0; p � 0.54; Fig. 10E].

DiscussionSpreading cortical hypoperfusion has been demonstrated duringthe early stage of migraine with aura and likely reflects a CSD-related vascular event (Ayata and Lauritzen, 2015), and decreasedcortical oxygenation has been suggested to trigger migraine head-ache (Schoonman et al., 2006; Arngrim et al., 2016). In our CSDrodent model, the cortical hypoperfusion and decreased tpO2

coincided with the emergence of the prolonged activation ofmeningeal afferents, suggesting a potential interaction. However,a key finding of this study was that despite exerting a powerfulinhibitory effect on the protracted cortical hypoperfusion and therelated decrease in oxygenation, naproxen did not ameliorate theactivation of meningeal afferents in response to CSD. Our datathus point to a dissociation between these CSD-related corticalmetabolic perturbations, or downstream processes, and the

Figure 6. Naproxen inhibits CSD-induced mechanical sensitization of meningeal afferents. A, B, Examples of experimental trials depicting the responses (post-stimulus time histogram; 0.1 s) oftwo C afferents to threshold (TH) and suprathreshold (STH) mechanical stimuli (green traces) applied to the dural receptive field during baseline recording and then at 30 min following CSD elicitationin control (A) and naproxen-treated animals (B). C–F, Note the lack of sensitization following naproxen treatment. Individual threshold and suprathreshold responses of sensitized and nonsensitizedafferents in control and naproxen-treated animals. G, H, Magnitudes of CSD-related threshold (G) and suprathreshold (H ) changes in mechanosensitivity depicting the inhibition of sensitization bynaproxen. p Values are based on a Mann–Whitney U test.

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Figure 7. Effects of the K(ATP) channel opener levcromakalim on CSD elicitation and CSD-evoked CBF changes. A, Levcromakalim increased basal CBF. B, C, Examples of the combined recordingof intracortical neural multiunit activity (MUA; post-stimulus time histogram; 1 s in middle panels) and CBF (bottom panels) showing a similar induction of CSD in control (B) and levcromakalim-treated animals (C). Note the initial depolarization (increased cortical activity; arrows) followed by the suppression and gradual recovery of neural activity. Also note the CSD-evoked corticalhyperemic response in the control animal and the diminished response in levcromakalim-treated animal (arrowheads). D, Time course changes of CBF in control and levcromakalim-treated animalsfollowing PP-induced CSD. Note that despite the decrease in CBF in levcromakalim-treated animals, values remained near predrug baseline values (denoted as 100%) unlike the marked decrease incontrol animals. E, CBF changes in control and levcromakalim-treated animals during the hyperemia stage. F, CBF changes during the hypoperfusion phase, 10 – 60 min following CSD. p Values arebased on a Mann–Whitney U test. PP, Pinprick.

Figure 8. Levcromakalim inhibits CSD-evoked cortical hypoxia. A, Time course changes (median and IQR) in cortical tpO2 values at baseline (BL) and during the acute hyperoxia and hypoxiaphases in response to CSD induction with pinprick (phases 1 and 2, respectively) in control and levcromakalim-treated animals. Note the shorter duration of the hypoxia phase followinglevcromakalim treatment. B, The prolonged, post-CSD decrease in tpO2 (phase 3) in control animals and its amelioration by levcromakalim are shown. Dashed line represents predrug baseline values.C–E, Differences between control and levcromakalim-treated animals in the hyperoxic response (AUC, phase 1; C), acute hypoxia (AUC, phase 2; D), and the prolonged decrease in tpO2 at 5–25 minpost-CSD (AUC, phase 3; E). p Values are based on a Mann–Whitney U test. Levcro, levcromakalim; PP, pinprick.

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mechanism responsible for driving menin-geal afferent activity. The ineffectiveness ofnaproxen was somewhat surprising in lightof its ability to reduce the persistent acti-vation of meningeal afferents followingapplication of a mixture of inflammatorymediators to their meningeal receptivefield (Levy et al., 2008)—a model of men-ingeal nociception (Strassman et al.,1996) and migraine pain (Oshinsky andGomonchareonsiri, 2007; Edelmayer etal., 2009; De Felice et al., 2013). This dis-crepancy suggests that the prolonged acti-vation of meningeal afferents in the wake ofCSD occurs via a distinct mechanism, onethat does not involve local nociceptiveactions of these classical inflammatory me-diators (Strassman et al., 1996) or vasocon-stricting prostanoids that mediate thecortical hypoperfusion following CSD (Shi-bata et al., 1992; Gariepy et al., 2017).

The cortical hyperemia that occursduring the CSD wave is likely driven inpart by the acute activation of meningealafferents, and the ensuing release of vaso-dilatory neuropeptides such as CGRP (Busijaet al., 2008). We found that naproxen also in-creased the initial cortical hyperemia,which likely mediated the conversion ofthe acute hypoxia phase to hyperoxia, butthese effects did not influence the acuteactivation of the afferents. These data sup-port the view that cortical vasodilationand its related increases in tissue oxygen-ation do not drive meningeal afferent activ-ity (Levy and Burstein, 2011), and thatduring the hyperemic phase of the CSDthere is a concurrent release of opposing va-soconstricting prostanoids.

During the prolonged cortical hypo-perfusion phase, CSD also gives rise topersistent vasodilatation of the middlemeningeal artery (Bolay et al., 2002), avascular response proposed to be medi-ated by prolonged activation of dural af-ferents and downstream signaling via acentral parasympathetic reflex (Bolay etal., 2002). Importantly, a recent study(Karatas et al., 2013) demonstrated thatthis CSD-evoked meningeal vasodilatoryresponse could be blocked by systemic ad-ministration of naproxen. That naproxendid not inhibit the CSD-evoked activationof meningeal afferents together with pre-vious data showing its inability to block parasympathetic outflowto the cranium (Akerman et al., 2012) suggests therefore thatCSD-evoked dural vasodilatation may not be driven by enhancedongoing meningeal afferent activity. Our present results thus callinto question the use of dural artery vasodilation as a surrogate ofincreased meningeal afferent activity and the activation of themigraine pain pathway in preclinical studies.

We found that counteracting the protracted cortical hypoper-fusion and prolonged decrease in tpO2 by preemptively increas-

ing CBF using levcromakalim was associated with inhibition ofthe acute and prolonged afferent activation phases. We propose,however, that the inhibitory effect of levcromakalim on theseafferent responses did not involve the normalization of CBF ortpO2 given that naproxen, which abrogated the cortical metabolicperturbations following CSD, failed to inhibit the afferent activa-tion. We cannot exclude the possibility that the cortical vasodi-lation and increased tpO2 induced by levcromakalim, beforethe onset of CSD, contributed somehow to the blockade of the

Figure 9. Levcromakalim inhibits CSD-evoked acute and prolonged meningeal afferent activation. A, B, Examples of single-unitrecording of an A-� afferent (A) and a C afferent (B), combined with laser Doppler flowmetry, depicting the lack of acute andprolonged afferents following the elicitation of CSD with a pinprick in animals treated with levcromakalim. C, In the presence oflevcromakalim, only one C afferent became acutely activated (open square outside the minimum/maximum box). D, Prolongedactivation was noted only in one A-� and three C afferents (open circle and squares outside the minimum/maximum box). PP,Pinprick.

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post-CSD afferent activation. However, the finding that levcro-makalim did not affect the basal ongoing activity of afferentssuggests that the vascular effects of levcromakalim were unlikelyto mediate to the inhibition of the CSD-evoked afferentactivation.

Cortical neurons also express K(ATP) channels (Karschin etal., 1997), and their prolonged activation by levcromakalim couldhave resulted in diminished cortical excitability (Soundarapand-ian et al., 2007), potentially affecting the elicitation of CSD. How-ever, levcromakalim did not inhibit the induction of CSD, andparticularly not the initial strong depolarization before the pro-longed suppression of cortical neural activity. K(ATP) activationin cortical neurons could, nonetheless, reduce the release of pro-nociceptive transmitters, such as glutamate (Zhao et al., 2013),

which may contribute to the acute activation, and potentially tothe prolonged activation, of meningeal afferents. Cortical astro-cytes also express K(ATP) channels (Thomzig et al., 2001), andtheir activation could potentially decrease the high extracellularK level that is attained during the passage of the CSD wave(Kraig and Nicholson, 1978) by enhancing K “spatial buffer-ing” (Kofuji and Newman, 2004). In response to CSD, elaboratedK in the cortical extracellular space has been suggested to dif-fuse into the leptomeninges and promote the acute activation ofmeningeal afferents and potentially also their prolonged activa-tion, at least in part (Pietrobon and Moskowitz, 2013; Zhao andLevy, 2017). Activation of astrocytic K(ATP) channels, leading todecreased parenchymal K, thus could have potentially inhibitedthe CSD-evoked afferent activation. Our recent finding that K-

Figure 10. Levcromakalim does not inhibit CSD-evoked mechanical sensitization of meningeal afferents. A, An example of an experimental trial depicting the CSD-evoked increase in mechanicalresponsiveness of a C afferent recorded from a levcromakalim-treated animal. B, C, Note that CSD-evoked sensitization at both the threshold and suprathreshold levels (poststimulus time histogram;0.1 s). Individual threshold (B) and suprathreshold (C) responses of sensitized and nonsensitized afferents. D, E, Comparisons between the magnitudes of CSD-related changes in mechanosensitivityat the threshold (D) and suprathreshold levels (E) depicting similar CSD-evoked sensitization responses in control and levcromakalim-treated animals. p Values are based on a Mann–Whitney U test.AU, Arbitrary units; Levcro, levcromakalim; STH, suprathreshold; TH, threshold.

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evoked acute activation of meningeal afferents was not associatedwith mechanical sensitization (Zhao and Levy, 2017) is also inagreement with the observation that levcromakalim did not in-hibit the CSD-evoked mechanical sensitization response. Finally,activation of K(ATP) channels expressed at the peripheral nerveendings of meningeal afferents could have also contributed to theinhibition of the afferent activation by reducing their overall ex-citability (Chi et al., 2007). However, the finding that levcro-makalim did not reduce basal ongoing discharge or inhibit themechanical sensitization argues against such a mechanism.

We found that naproxen distinctly inhibited the CSD-evokedmechanical sensitization of meningeal afferents. That levcromaka-lim treatment greatly reduced the acute hypoxia phase and nor-malized the prolonged cortical hypoperfusion and decreasedtpO2, but did not inhibit the enhanced mechanical responsive-ness, suggests, however, that the mechanism underlying the me-chanical sensitization following CSD is unrelated to the changesin cortical perfusion and tpO2. It should be noted that whilelevcromakalim treatment normalized CBF and tpO2 (to valuesnot different than those observed before CSD), it did not inhibitthe process that led to their decreases following CSD. We there-fore propose that the mediators that contribute to these meta-bolic changes (likely cyclooxygenase derived; see below) areresponsible for the afferent sensitization rather than the corticalhypoperfusion and abnormally reduced tpO2 per se. It is possiblethat the acute hypoxic phase that occurred in levcromakalim-treatedanimals (albeit for only a fraction of the time noted in controlanimals) may have contributed somehow to the afferent sensiti-zation process. However, we also observed brief cortical hypoxiain some naproxen-treated animals, further suggesting a dissoci-ation between the acute hypoxic phase and the afferent changes.

The discrepancy between the effects of naproxen and levcro-makalim on the CSD-evoked afferent activation and mechanicalsensitization responses supports the view that these two migraine-related nociceptive responses are mediated by distinct mecha-nisms (Levy and Strassman, 2004; Zhang et al., 2011a, 2013; Zhaoand Levy, 2016). The site of action of naproxen and the nature ofthe cyclooxygenase-derived factors that distinctly promote thesensitization of meningeal afferents following CSD require fur-ther studies. The release of cyclooxygenase-derived sensitizingmediators from cortical neurons and glial limitans astrocytes thatinterface with the meninges (Xu et al., 2004) could contribute tothe afferent sensitization. The mode of cortex-to-meninges trans-port of cyclooxygenase-derived prostanoids could involve diffu-sion or bulk flow via the glymphatic system (Iliff et al., 2012;Schain et al., 2017). The transport of sensitizing factors from theparenchymal interstitial space into the subarachnoid CSF couldinfluence primarily leptomeningeal afferents and meningeal af-ferents that terminate within arachnoid granulations (vonDuring and Andres, 1991; Fricke et al., 1997). Additional flow ofsensitizing mediators from the subarachnoid CSF into the durallymphatics system (Raper et al., 2016; Louveau et al., 2017) couldaffect the responses of dural afferents. Naproxen treatment couldhave also inhibited the CSD-evoked meningeal afferent sensitiza-tion by targeting constitutively expressed meningeal cyclooxy-genase in meningeal vascular and immune cells (Zhang et al.,2009) and the ensuing release of sensitizing prostanoids (Zhanget al., 2011a).

In summary, our findings point to a dissociation between theCSD-evoked cortical metabolic perturbations and the prolongedactivation and mechanical sensitization of meningeal afferents.The findings that K(ATP) activation and cyclooxygenase inhibi-tion have distinct effects on the CSD-evoked activation and sen-

sitization of meningeal afferents may explain the ineffectivenessof cyclooxygenase inhibitors in many cases of migraine (Suthisi-sang et al., 2010). A combination therapy, which includes acyclooxygenase inhibitor and a K(ATP) channel opener, may beexplored for treating the headache in migraine with aura.

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