Flow cytometric analysis of BrdU incorporation as a high-throughput method for measuring adult neurogenesis in the mouse
Flow cytometric analysis of BrdU incorporation as a high-throughput method for measuring adult neurogenesis in the mouse
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DOI：
10.1016/j.vascn.2008.12.002
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发表时间：
2009-03-01
影响因子：
1.9
通讯作者：
Lucki, Irwin
中科院分区：
文献类型：
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作者：
Balu, Darrick T.;Hodes, Georgia E.;Lucki, Irwin
新神经元的产生发生在整个成年期的离散脑区，并可能受到神经精神疾病和治疗性药物治疗的调节。目前研究这一过程的大多数方法是使用免疫组织化学方法对新生细胞进行5-溴-2-脱氧尿苷（BrdU）标记，然后对BrdU阳性细胞进行显微镜计数。这种方法是耗时和劳动密集型的，通常需要几个星期的时间来分析。因此，我们研究了一种利用流式细胞术在体内测量成年小鼠海马中BrdU掺入的方法，这种方法通常可以在一天内分析数据。结果：本研究比较了多种BrdU给药和加载方案，以确定产生最佳信噪比的给药策略。还比较了BrdU在不同脑区的掺入情况。该方法对许多实验性疾病操作很敏感。1型糖尿病的诱导和去甲肾上腺素的消耗减少了海马细胞的增殖。相反，长期的电休克治疗，抑郁症的躯体治疗，以及抗抑郁药物氟西汀的慢性治疗会增加海马细胞的增殖。氟西汀治疗后的第14天就检测到细胞增殖的增加。此外，在同一动物体内，用免疫组织化学方法和流式细胞术方法比较细胞增殖的测量结果是收敛的，并且彼此之间存在显著的相关性。流式细胞术也有足够的灵敏度来量化新生细胞的存活率。讨论：这些实验验证了流式细胞术在分析海马细胞增殖和存活方面的可靠性和高通量的实用性。流式细胞术提供的快速分析使其在新药物发现和生理学中得到利用。(C) 2009爱思唯尔公司版权所有。
Introduction: The generation of new neurons occurs throughout adulthood in discrete brain regions, and may be regulated by neuropsychiatric diseases and therapeutic drug treatments. Most current methods that study this process measure the labeling of newborn cells by 5-bromo-2-deoxyuridine (BrdU) using immunohistochemical methods followed by the microscopic counting of BrdU positive cells. This method is time consuming and labor intensive, typically taking several weeks to analyze. Methods: Therefore, we characterized a method to measure BrdU incorporation in the adult mouse hippocampus in vivo by using flow cytometry, which normally allows analysis of data within a single day. Results: The present study compared multiple BrdU dosing and loading protocols to determine a dosing strategy that produced the best signal to noise ratio. BrdU incorporation was also compared across different brain regions. The method was sensitive to a number of experimental disease manipulations. Induction of type-1 diabetes and depletion of norepinephrine reduced hippocampal cell proliferation. In contrast, chronic administration of electroconvulsive shock, a somatic treatment for depression, as well as chronic treatment with the antidepressant fluoxetine elevated hippocampal cell proliferation. This increase in cell proliferation with fluoxetine was detected as early as 14 days into treatment. Moreover, comparing measures of cell proliferation obtained by immunohistochemical and flow cytometric methods within the same animals were convergent and significantly correlated to each other. Flow cytometry was also sufficiently sensitive to quantify the survival of newly born cells. Discussion: These experiments validate the utility of flow cytometry in analyzing hippocampal cell proliferation and survival in a reliable and high-throughput fashion. The speedy analysis afforded by flow cytometry lends itself to be utilized in novel drug discovery and physiology. (C) 2009 Elsevier Inc. All rights reserved.