FAAH inhibition in migraine pain

This dataset comprises the findings obtained in the study aimed at investigating the relevance of fatty-acid amide hydrolase (FAAH) inhibition in the nitroglycerin (NTG) animal model of migraine, by testing the effects of the globally active FAAH inhibitor URB597. The attention was focused on mapping neuronal c-Fos protein expression, measurement of anandamide (AEA) and palmitoylethanolamide (PEA) levels in brain areas and in trigeminal ganglia, evaluation of pain-related trigeminal behavior and quantification of molecular mediators in rats that received URB597 (2 mg/kg i.p.) either before or after NTG administration (10 mg/kg, i.p.).

Evaluation of c-Fos expression by immunohistochemistry: cell counts of individual brain nuclei were made from every sixth section throughout their rostrocaudal extent for each rat. The nuclei evaluated were identified with the rat brain atlas Paxinos and Watson 4th edition. Trigeminal nucleus caudalis (TNC): from bregma −14.08 mm (interaural −5.08 mm) until −17 mm (interaural −8 mm); nucleus tractus solitarii (NTS): from bregma −13.24 mm (interaural −4.24 mm) until −13.68 mm (interaural −4.68 mm); locus ceruleus (LC): from bregma −9.80 mm (interaural −0.80 mm) until −10.04 mm (interaural −1.04 mm); parabrachial nucleus (PAB): from bregma −8.80 mm (interaural 0.20 mm) until −9.80 mm (interaural −0.50 mm); periaqueductal gray (PAG): from bregma −7.80 mm (interaural 1.20 mm) until −8.80 mm (interaural 0.20 mm).

Measurement of AEA and PEA levels: medulla, cervical spinal cord and trigeminal ganglia were homogenized in cold methanol (2 ml) containing AEA-d4 and PEA-d4 as internal standards. After extraction the lipids were measured using a Xevo TQ UPLC-MS/MS system equipped with a reversed-phase BEH C18 column (2.1 × 50 mm, 1.7 μm particle size) (Waters, Milford, USA).

Pain-related behavior in the orofacial formalin test: the face rubbing was measured counting the seconds the animal spent grooming the injected area (upper lip, lateral to the nose) with the ipsilateral forepaw or hindpaw 0–6 min (Phase I) or 12–45 min (Phase II) after formalin injection (50 µl, s.c.). The observation time was divided into 15 blocks of 3 min each.

mRNA expression levels: calcitonin gene-related peptide (CGRP), neuronal nitric oxide synthase (nNOS), interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-alpha) mRNA were evaluated in in medulla, cervical spinal cord and trigeminal ganglia; whereas, in dura mater were evaluated IL-1β and inducible NOS (iNOS). mRNA levels were measured by rt-PCR. All samples were assayed in triplicate and gene expression levels were calculated according to 2−∆∆Ct = 2− (∆Ct gene − ∆Ct housekeeping gene) formula by using Ct (cycle threshold) values.

本数据集包含一项研究的所得结果，该研究旨在探究脂肪酸酰胺水解酶（fatty-acid amide hydrolase, FAAH）抑制作用在偏头痛硝酸甘油（nitroglycerin, NTG）动物模型中的相关性，通过测试全球活性FAAH抑制剂URB597的干预效果。研究重点包括绘制神经元c-Fos蛋白表达图谱、检测脑区与三叉神经节中的花生四烯酸乙醇胺（anandamide, AEA）和棕榈酰乙醇酰胺（palmitoylethanolamide, PEA）水平、评估疼痛相关三叉神经行为，以及对在NTG给药前或给药后腹腔注射（intraperitoneal, i.p.）URB597（2 mg/kg）的大鼠体内的分子介质进行定量分析，其中NTG给药剂量为10 mg/kg，腹腔注射。

免疫组化法检测c-Fos蛋白表达：每只大鼠取其沿吻尾方向全范围的脑切片中每6张取1张，统计各脑核的细胞数量。所评估的脑核参照《大鼠脑图谱》（Paxinos与Watson第4版）进行定位：三叉神经尾侧核（trigeminal nucleus caudalis, TNC）：位于前囟（bregma）-14.08 mm（耳间线-5.08 mm）至-17 mm（耳间线-8 mm）区域；孤束核（nucleus tractus solitarii, NTS）：位于前囟-13.24 mm（耳间线-4.24 mm）至-13.68 mm（耳间线-4.68 mm）区域；蓝斑（locus ceruleus, LC）：位于前囟-9.80 mm（耳间线-0.80 mm）至-10.04 mm（耳间线-1.04 mm）区域；臂旁核（parabrachial nucleus, PAB）：位于前囟-8.80 mm（耳间线0.20 mm）至-9.80 mm（耳间线-0.50 mm）区域；中脑导水管周围灰质（periaqueductal gray, PAG）：位于前囟-7.80 mm（耳间线1.20 mm）至-8.80 mm（耳间线0.20 mm）区域。

AEA与PEA水平检测：将延髓、颈髓和三叉神经节置于含氘代花生四烯酸乙醇胺（AEA-d4）和氘代棕榈酰乙醇酰胺（PEA-d4）作为内标的2 ml冰冷甲醇中进行匀浆。脂质提取完成后，采用配备反相BEH C18色谱柱（2.1 × 50 mm，粒径1.7 μm）的Xevo TQ超高效液相色谱-串联质谱（UPLC-MS/MS）系统进行检测（沃特世公司，美国米尔福德）。

口面部福尔马林试验疼痛相关行为评估：记录大鼠在福尔马林注射（50 µl，皮下注射（subcutaneous, s.c.））后0~6 min（第一阶段）与12~45 min（第二阶段）内，用同侧前爪或后爪擦拭注射部位（鼻外侧上方唇）的时长。观察时间被划分为15个时长为3 min的时间段。

mRNA表达水平检测：在延髓、颈髓和三叉神经节中检测降钙素基因相关肽（calcitonin gene-related peptide, CGRP）、神经元型一氧化氮合酶（neuronal nitric oxide synthase, nNOS）、白细胞介素-1β（interleukin-1β, IL-1β）和肿瘤坏死因子-α（tumor necrosis factor-alpha, TNF-α）的mRNA表达水平；在硬脑膜中则检测IL-1β与诱导型一氧化氮合酶（inducible NOS, iNOS）的mRNA表达水平。mRNA水平通过rt-PCR进行检测，所有样本均设置3次重复，采用循环阈值（Ct）值，按照公式2^(-ΔΔCt) = 2^(-(ΔCt基因 - ΔCt管家基因))计算基因表达水平。