Fairbanks Stacy L, Vest Rebekah, Verma Saurabh, Traystman Richard J, Herson Paco S
Department of Anesthesiology, University of Colorado School of Medicine.
J Vis Exp. 2013 Dec 9(82):e50758. doi: 10.3791/50758.
Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.
长期以来，人们一直观察到神经元对缺血性损伤和神经退行性疾病的易感性存在性别差异，但导致这些差异的信号传导机制仍不清楚。原代解离胚胎神经元培养提供了一个简化的实验模型，用于研究缺血或疾病导致细胞死亡过程中涉及的神经元细胞信号传导；然而，如今研究中使用的大多数神经元培养物都是混合性别的。研究人员能够且确实在神经元损伤和疾病模型的混合性别神经元培养物中测试性类固醇治疗的效果，但越来越多的证据表明，雌性大脑对雄激素、雌激素和孕酮的反应与雄性大脑不同。此外，新生雄性和雌性啮齿动物对缺血性损伤的反应也不同，雄性在脑缺血后比雌性经历更严重的损伤。因此，混合性别的神经元培养物可能会掩盖和混淆实验结果；重要信息可能会被遗漏。出于这个原因，科罗拉多大学医学院的赫尔森实验室通常从海马体和皮质制备性别分层的原代解离胚胎神经元培养物。在收获脑组织之前对胚胎进行性别鉴定，将雄性和雌性组织分别解离、分别接种并分别培养。使用这种方法，赫尔森实验室已经证明离子通道TRPM2在缺血性细胞死亡中具有雄性特异性作用。在本手稿中，我们分享并讨论了对胚胎小鼠进行性别鉴定以及制备性别分层海马体原代解离神经元培养物的方案。该方法可适用于制备性别分层的皮质培养物，胚胎性别鉴定方法可与其他方案结合使用，用于任何认为性别是结果重要决定因素的研究。
