在Cre/loxP基因重组小鼠脑内植入“光控开关”建立偏头痛模型

The lack of reliable animal models has been the very obstacle of studying the mechanisms of migraine and the effect of acute and phylactic migraine drugs. Although the knock-in mice with the familial hemiplegic migraine genes became the hoping tools for migraine studying, the corresponding ion channels, which distribute all over the system, express specifically in different organs or tissue with different level. Most of the homozygous knock-in mice with FHM mutations die at birth or on postnatal day, while the heterozygous mice with FHM mutations do not exhibit an overt phenotype. It is important and urgent to develop more reliable migraine animal model. In our study, based on the theory that migraine is a disorder of brain excitatory-inhibitory balance, we plan to develop two types of mouse model, involving the combination of optogenetics and Cre/loxP genetic recombination. On one side, we are going to induce bi-stable opsin ChR2-SSFO into two types of neurons in different brain regions of two lines of Cre- recombinase mice, to activate or inactivate neural activity by manipulating the opsins with light in millisecond precision, in order to develop an increasing susceptibility model of migraine. On the other side, a conditional genetic knock-in mouse model with FHM3 mutation (Q1489K) will be developed, involving the loxP recombination technique. A ChR2 （H143R）opsin will be transplanted at the same time, hoping that the opsin will stabilize the excitatory/inhibitory imbalance to prolong the survival life of the mutant mice, which will be a model of chronic spontaneous migraine attacks. We hope that both of the new animal models can be very efficient and powerful for the studying of migraine in the future.

缺少可靠的动物模型是偏头痛研究的难点。虽然家族性偏瘫型偏头痛（familial hemiplegic migraine, FHM）致病基因敲入（knock-in，KI）小鼠是带来希望的工具，但FHM基因编码的离子通道既在全身广泛分布又有高度组织特异性，多数致病基因的纯合KI小鼠死于胚胎期，而杂合动物的偏头痛表现不明显，造模方法仍需改良。本项目以偏头痛的兴奋/抑制稳态失衡学说为依据，一方面将光敏蛋白引入几种标记不同类型神经元的Cre重组酶小鼠中，通过直接光控特定脑区某类神经元活性，构建偏头痛易感性增强小鼠模型；另一方面利用Cre/loxP基因重组技术培育FHM3型基因条件性敲入小鼠，对其植入光敏蛋白，光控调节其神经元活性来缓解基因突变所致脑内兴奋/抑制稳态失衡，以构建慢性自发性偏头痛小鼠模型。从而为更加深入研究偏头痛发病机制，为新药研发提供更为有效、可靠、更接近偏头痛临床表现的动物模型。

本项目旨在建立一个更为可靠的、标准化的、接近临床偏头痛发作的偏头痛疾病动物模型。项目立项伊始，我们利用光遗传学技术和化学遗传技术结合条件敲除转基因小鼠，拟构建自发性偏头痛小鼠模型。在初步研究中，我们发现如果抑制中脑导水管周围灰质中的PV+神经元，小鼠的头痛行为减少，提示调节上述神经元或可起到镇痛作用。但由于我们采用的头痛刺激方式可重复性差，这一发现至今未得到肯定。为此，我们在后续研究中重点进行清醒状态下小鼠头痛模型的标准化构建。我们利用改进的电刺激器制作电刺激小鼠上矢状窦旁硬脑膜模型，动物在清醒状态下明确出现头痛相关行为增多，且还出现了畏光这一偏头痛特有行为的增多。上述行为在给予特异性偏头痛急性期药物舒马曲坦后能后部分抑制。且在三叉神经脊束核等头痛相关脑区内，模型小鼠的c-Fos阳性细胞表达增多，进一步提示该模型所具有明显的头痛模型相关特征。电刺激模型模拟了无先兆偏头痛的类似表现，我们使用KCl注射视皮层诱发皮层扩布性抑制（CSD）来模拟先兆偏头痛的表现。既往这类研究均在动物麻醉状态下进行，并不确定CSD一定能够诱发头痛相关行为的出现。但我们不仅成功诱发出血流量扩布减少的CSD特征，还观察到清醒状态下给予CSD刺激动物出现明确的头痛相关行为的增多，且在三叉神经脊束核区的c-Fos表达增多。上述情况仍然能够被舒马曲坦部分抑制，提示清醒状态下向视皮层注射KCl头痛动物模型培育成功。我们在培育这两个动物模型的同时，研发了小鼠观察箱等相关实验动物疼痛行为观察工具。在一定程度上帮助我们标准化、客观量化动物的行为学指标。我们在上述模型基础上进行了神经影像学研究发现，给予低频头痛刺激，动物的三叉神经血管复合体区的白质体积明显增加，进一步提示我们下一步工作的重点是利用光遗传学、化学遗传学技术手段操作上述区域的神经细胞，尝试培育出自发性头痛动物模型。在本项目资助下，我们发表SCI论文11篇，申请专利3项，获得专利授权2项。