Estradiol and Zoster Associated Orofacial Pain

摘要 带状疱疹(HZ)或“带状疱疹”患者可能会遭受口腔面部疼痛的折磨。口腔面部HZ是由 三叉神经节内潜伏的水痘带状疱疹病毒(VZV)重新激活。重要的是，女性报告HZ疼痛 发病率是男性的三倍，但造成这种性别差异的机制尚不清楚。在我们实验室的屏幕上 在口面部疼痛通路的五个不同区域的20,000多个基因显示，最大的变化是 基因表达发生在丘脑，如谷氨酸脱羧酶2(GAD2)和囊泡GABA 转运蛋白(VGAT)在动情前期(高17β-雌二醇，E_2)升高，但不在动情间期(低E_2)升高。在……里面 初步研究表明，雌性大鼠有更大的VZV诱导的口面部伤害性反应 与男性相比；反映了人类的性别差异。我们还表明，减少雌激素2的产生 雄性和雌性大鼠增加其伤害性反应，并最终下调VGAT在大脑中的表达 丘脑增加了伤害性反应。基于这些研究，我们假设雌二醇会减弱 增加GAD2或VGAT，增强GABA能神经抑制的口面部伤害性 丘脑，以减轻疼痛。为了验证这一假设，目标1将描述丘脑对VZV诱导的VZV的控制 口腔面部伤害性反应。Aim 1的工作假设是GABA中间神经元抑制神经元 减少丘脑VZV引起的伤害性反应。为了首先验证这一假设，VZV诱导了伤害性感受 减弱抑制性中间神经元后，记录神经元的活动。第二，伤害性和 在调节这些中间神经元中GAD2和VGAT的表达后，神经元的活性将被确定。目标 2将确定性激素在调节VZV诱导的伤害性反应中的作用。我们的工作 假设E2会增加丘脑中VGAT和GAD2的表达，导致 神经元活动和伤害性反应。为了首先检验这一假设，伤害性反应和 雌性和雄性大鼠在注射E2或减少E2产生后，将测量神经元的活动。 其次，在GAD2和VGAT表达下调后，将测量伤害性感受和神经元活动 在雌激素水平不同的大鼠中。目标3将特征性类固醇调节VGAT的机制 和GAD2影响VZV诱导的口面部伤害性反应。我们的工作假说是E2结合 雌激素受体α(ERα)引起丘脑VGAT和GAD2表达增加 在伤害性感受的减弱方面。为了测试这一想法，伤害性反应和神经元活动将 用CRISPR/CAS9技术突变ERα启动子区后的大鼠定量。从这三个目标出发 我们期望确定1)GABA中间神经元抑制丘脑信号以减轻口面部疼痛，2) GAD2和VGAT通过ERα依赖机制增加，3)GAD2和VGAT VGAT对观察到的HZ疼痛的性别差异负有部分责任。确定一种机制，通过该机制 E2影响HZ相关疼痛将为治疗这种慢性疾病的患者提供新的靶点。

ABSTRACT People with herpes zoster (HZ) or “shingles” can suffer from orofacial pain. Orofacial HZ is caused by the reactivation of latent varicella zoster virus (VZV) in the trigeminal ganglia. Importantly, women report HZ pain three times more often than men, but the mechanism for this sex disparity is unknown. A screen by our lab of over 20,000 genes in five different regions of the orofacial pain pathway revealed that the greatest change in gene expression occurred in the thalamus, such that glutamate decarboxylase 2 (GAD2) and vesicular GABA transporter (VGAT) were elevated at proestrus (high 17 β-estradiol, E2) but not diestrus (low E2). In preliminary studies we showed that female rats have a greater VZV induced orofacial nociceptive response in comparison to males; mirroring the human sex difference. We also showed that reducing E2 production in male and female rats increases their nociceptive response and lastly, knock-down of VGAT expression in the thalamus increased the nociceptive response. Based on these studies we hypothesized that E2 attenuates orofacial nociception by increasing GAD2 or VGAT, enhancing GABAergic neural inhibition within the thalamus to reduce pain. To test this hypothesis, Aim 1 will characterize thalamic control of the VZV induced orofacial nociceptive response. The working hypothesis for Aim 1 is that GABA interneurons inhibit neurons in the thalamus to reduce VZV induced nociception. To test this hypothesis first, VZV induced nociception and neuronal activity will be recorded after attenuating inhibitory interneurons. Second, nociception and neuronal activity will be determined after modulating GAD2 and VGAT expression in these interneurons. Aim 2 will determine the role of sex steroids in modulating the VZV induced nociceptive response. Our working hypothesis is that E2 will increase expression of VGAT and GAD2 in the thalamus causing attenuation of neuronal activity and the nociceptive response. To test this hypothesis first, the nociceptive response and neuronal activity will be measured in female and male rats after administering E2 or reducing E2 production. Second, nociception and neuronal activity will be measured after knock-down of GAD2 and VGAT expression in rats with varied E2 levels. Aim 3 will characterize the mechanism by which sex steroids modulate VGAT and GAD2 to affect the VZV induced orofacial nociceptive response. Our working hypothesis is that E2 binds to estrogen receptor alpha (ERα) causing increased expression of VGAT and GAD2 in the thalamus resulting in attenuation of nociception. To test this idea, the nociceptive response and neuronal activity will be quantitated in rats after mutating ERα promoter sites using CRISPR/Cas9 technology. From these three aims we expect to determine 1) that GABA interneurons inhibit thalamic signaling to reduce orofacial pain, 2) that GAD2 and VGAT increases in these neurons through an ERα dependent mechanism, and 3) that GAD2 and VGAT are responsible, in part, for the sex difference observed in HZ pain. Identifying a mechanism by which E2 affects HZ-associated pain will provide new targets for treating people suffering from this chronic disorder.