Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, KS, USA.
Headache. 2011 May;51(5):674-92. doi: 10.1111/j.1526-4610.2011.01882.x.
The objectives of this study were to develop a preclinical rodent model that produces migraine-like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of calcitonin gene-related peptide (CGRP) and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors.
Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (phosphate-buffered saline [PBS]) to the dura were measured to model changes in routine activity and allodynia. CGRP, an important mediator of migraine pathogenesis, and the 3 components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura.
Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 µL volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 minutes post-application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real-time polymerase chain reactions were used to measure the expression of amplified constructs using gene-specific primers for CGRP, RAMP1, CLR, and RCP.
Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µL IS groups), and males showed increased perimasseter withdrawal responses (20 µL IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP-encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP-encoding mRNAs. In the medulla, females had higher baseline levels of CGRP-encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP-encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP, and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP-related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP-related genes were upregulated by both IS and PBS applications.
This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine-like behaviors occur after meningeal inflammation.
本研究旨在开发一种基于国际头痛协会诊断标准的产生偏头痛样行为的啮齿动物临床前模型，确定是否存在性别差异，并确定三叉神经节或延髓中降钙素基因相关肽 (CGRP) 及其受体编码基因的表达是否与这些行为相关。
很少有偏头痛的动物研究测试与偏头痛诊断标准相关的行为。在这项研究中，通过向硬脑膜施加炎症汤 (IS) 或载体 (磷酸盐缓冲盐水 [PBS]) 来测量面部区域活动和机械敏感性的变化，以模拟常规活动和痛觉过敏的变化。CGRP 是偏头痛发病机制的重要介质，其 3 个受体成分，降钙素样受体 (CLR)、受体活性修饰蛋白 1 (RAMP1) 和受体成分蛋白 (RCP) 的 mRNA 在三叉神经节和延髓中进行了定量，以确定基线性别差异和与 IS 或 PBS 向硬脑膜应用相关的变化。
雄性和雌性 Sprague-Dawley 大鼠被植入硬脑膜套管。将大鼠分为接受 10 或 20 μL 体积 IS 或 PBS 的组。在手术前和术后 7 天进行基线行为测试，并进行 8 次重复硬脑膜应用。使用压力板活动计进行期间和之后的运动活动评估，以提供有关行进距离、低移动性发作、空间限制和集中能量的信息。应用后 20 分钟进行眶周和眶下机械测试，以测量痛觉过敏。最后一次硬脑膜处理后 30 分钟处死大鼠，从同侧三叉神经节和同侧延髓中分离出总 RNA。使用基因特异性引物进行定量实时聚合酶链反应，以测量 CGRP、RAMP1、CLR 和 RCP 扩增构建物的表达。
男性和女性都表现出 IS 应用的行为效应，但存在明显的性别差异。女性在较低剂量下表现出作用，并且活动变化持续时间更长，但男性需要更少的 IS 应用才能表现出行为变化。女性表现出眶周和眶下机械测试的撤回反应增加（10 μL IS 组），而男性表现出眶下肌撤回反应增加（20 μL IS 组）。在三叉神经节中，CGRP 编码 mRNA 没有基线性别差异，但女性的 RAMP1、CLR 和 RCP 编码 mRNA 基线表达较低。在延髓中，女性的 CGRP 编码 mRNA 基线水平较高，RAMP1、CLR 和 RCP 编码 mRNA 基线水平较低。IS 和 PBS 均增加了雄性三叉神经节中 CGRP、RAMP1、RCP 和 CLR 编码 mRNA 的表达，但在雌性中，只有 CLR 和 RCP 增加。在延髓中，IS 和 PBS 均增加了雄性 CGRP、CLR 的表达，以及雌性 CLR 和 RCP 的表达。因此，CGRP 相关基因的表达并未反映 IS 和 PBS 组之间的行为差异。相反，IS 和 PBS 应用均可上调 CGRP 相关基因的表达。
本研究表明，向硬脑膜施加 IS 会导致与 IS 相关的运动活动和面部痛觉过敏显著变化，以及显著的性别差异，表明国际头痛协会诊断标准可用于设计偏头痛的啮齿动物行为模型。此外，在三叉神经节和延髓中，CGRP 及其受体的表达均存在明显的基线性别差异，但 CGRP 和其受体的大多数表达变化均存在于 IS 和 PBS 处理的大鼠中。这表明 CGRP 途径对颅内压或脑膜伸展的变化有反应，而偏头痛样行为发生在脑膜炎症之后。
