Peripheral mechanisms of primary headaches such as a migraine remain unclear. Meningeal afferents surrounded by multiple mast cells have been suggested as a major source of migraine pain. Extracellular ATP released during migraine attacks is a likely candidate for activating meningeal afferents via neuronal P2X receptors. Recently, we showed that ATP also increased degranulation of resident meningeal mast cells ( Nurkhametova et al., 2019 ). However, the contribution of ATP-induced mast cell degranulation in aggravating the migraine pain remains unknown. Here we explored the role of meningeal mast cells in the pro-nociceptive effects of extracellular ATP. The impact of mast cells on ATP mediated activation of peripheral branches of trigeminal nerves was measured electrophysiologically in the dura mater of adult wild type (WT) or mast cell deficient mice. We found that a spontaneous spiking activity in the meningeal afferents, at baseline level, did not differ in two groups. However, in WT mice, meningeal application of ATP dramatically (24.6-fold) increased nociceptive firing, peaking at frequencies around 10 Hz. In contrast, in mast cell deficient animals, ATP-induced excitation was significantly weaker (3.5-fold). Application of serotonin to meninges in WT induced strong spiking. Moreover, in WT mice, the 5-HT3 antagonist MDL-7222 inhibited not only serotonin but also the ATP induced nociceptive firing. Our data suggest that extracellular ATP activates nociceptive firing in meningeal trigeminal afferents via amplified degranulation of resident mast cells in addition to direct excitatory action on the nerve terminals. This highlights the importance of mast cell degranulation via extracellular ATP, in aggravating the migraine pain.

Background and Purpose-Perivascularly positioned cerebral mast cells (MC) have been shown to participate in acute blood-brain barrier disruption and expansive brain edema following experimental transient cerebral ischemia. However, the underlying molecular mechanisms remain unknown. Because proteolytic gelatinase enzymes, matrix metalloproteinases (MMP)-2 and MMP-9, are thought to have a central role in compromising the integrity of the blood-brain barrier following ischemia, we examined whether cerebral MCs influence gelatinase activity in ischemic cerebral microvasculature. Methods-Rats underwent 60 minutes of middle cerebral artery occlusion followed by 3-hour reperfusion, and were treated with a MC-stabilizing (cromoglycate), or Key Words: mast cells Ⅲ gelatinases Ⅲ blood-brain barrier E xpansive brain edema is a frequent cause of death after large ischemic strokes, 1 as it reduces blood flow in the ischemic penumbra and can lead to herniation of brain structures. Edema is predominantly caused by functional and structural disturbance of the blood-brain barrier (BBB). Proteolytic gelatinase enzymes, most importantly matrix metalloproteinases (MMP)-2 and MMP-9, are considered to be central mediators of ischemic BBB disruption; this is because of their ability to degrade components of microvascular basal lamina, especially collagen type IV, 2-4 and to disrupt tight junction proteins. 5 The mechanisms triggering early activation of gelatinases in ischemic brain remain under active debate. 2,6 Mast cells (MC) are resident inflammatory cells positioned in the outer layers of various tissues (eg, mucosa, epithelia, and vasculature) in mammals, including the central nervous system. 7 We and others have previously shown that MCs have a role in the pathophysiology of ischemic, hypoxic-ischemic, and hemorrhagic brain injury. 8 -11 Both pharmacological inhibition and genetic deficiency of MCs led to a significant reduction in postischemic BBB disruption, cerebral edema, and neutrophil infiltration, 8 presenting MC inhibition as a potential therapeutic avenue to prevent inflammatory damage to the neurovasculature, especially in conjunction with thrombolytics. 9 We envisaged a molecular pathway for ischemic BBB disruption where factors liberated from MCs might fuel gelatinase activation within the targeted neurovascular unit and lead to degradation of the microvascular wall, especially Materials and Methods AnimalsThe in vivo methodology has been described in detail previously. 8 Briefly, adult, male, Wistar rats (Harlan Nederland) and WsRc Ws/Ws rats (Japan SLC, Inc), 290 to 340g, were anesthetized with ketamin hydrochloride (intraperitoneal, 50 mg/kg, Ketalar, Parke-Davis) and medetomidine hydrochloride (subcutaneous, 0.5 mg/kg, Domitor, Orion). The left femoral artery and vein were cannulated for measurements of blood pressure, arterial pH, blood gases, and blood glucose and drug and/or vehicle infusions. Cerebral blood flow was measured on-line with laser-Doppler flowmetry (Oxy-Flow, Oxford Optronix) as described...

Background Plasma glial fibrillary acidic protein (GFAP) and tau are promising markers for differentiating acute cerebral ischemia (ACI) and hemorrhagic stroke (HS), but their prehospital dynamics and usefulness are unknown. Methods We performed ultra-sensitivite single-molecule array (Simoa®) measurements of plasma GFAP and total tau in a stroke code patient cohort with cardinal stroke symptoms [National Institutes of Health Stroke Scale (NIHSS) ≥3]. Sequential sampling included 2 ultra-early samples, and a follow-up sample on the next morning. Results We included 272 cases (203 ACI, 60 HS, and 9 stroke mimics). Median (IQR) last-known-well to sampling time was 53 (35–90) minutes for initial prehospital samples, 90 (67–130) minutes for secondary acute samples, and 21 (16–24) hours for next morning samples. Plasma GFAP was significantly higher in patients with HS than ACI (P < 0.001 for <1 hour and <3 hour prehospital samples, and <3 hour secondary samples), while total tau showed no intergroup difference. The prehospital GFAP release rate (pg/mL/minute) occurring between the 2 very early samples was significantly higher in patients with HS than ACI [2.4 (0.6–14.1)] versus 0.3 (−0.3–0.9) pg/mL/minute, P < 0.001. For cases with <3 hour prehospital sampling (ACI n = 178, HS n = 59), a combined rule (prehospital GFAP >410 pg/mL, or prehospital GFAP 90–410 pg/mL together with GFAP release >0.6 pg/mL/minute) enabled ruling out HS with high certainty (NPV 98.4%) in 68% of patients with ACI (sensitivity for HS 96.6%, specificity 68%, PPV 50%). Conclusions In comparison to single-point measurement, monitoring the prehospital GFAP release rate improves ultra-early differentiation of stroke subtypes. With serial measurement GFAP has potential to improve future prehospital stroke diagnostics .