Determination of true Glia-Neuron Ratios: Validation of the Isotropic Fractionato

真实胶质细胞-神经元比率的测定：各向同性分数的验证

• 批准号: 8725753
• 负责人: CHRISTOPHER S VON BARTHELD
• 金额: $ 7.1万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8725753
• 关键词: Animals; Antibodies; Astrocytes; Autistic Disorder; Basic Science; Belief; Biological Models; Bipolar Depression; Bipolar Disorder; Brain; Brain region; Calibration; Cell Count; Cell Density; Cell Nucleus; Cells; Chickens; Clinical Research; Conflict (Psychology); Confusion; Corpus Callosum; DNA; Data; Disease; Disputes; Endothelial Cells; Frequencies; Gold; Health; Histology; Human; Journals; Knowledge; Label; Language; Lead; Measures; Mental Depression; Mental disorders; Methods; Microglia; Neuroglia; Neurons; Neurosciences; Oligodendroglia; Optic Nerve; Optics; Paper; Patients; Ploidies; Publishing; Recovery; Reporting; Sampling; Schizophrenia; Schools; Science; Series; Solutions; Structure; Techniques; Textbooks; Ursidae Family; Validation; Work; base; cell type; density; design; evidence base; frontal lobe; gray matter; interest; morphometry; nervous system disorder; nonhuman primate; novel; novel strategies; particle; public health relevance; tool; white matter

DESCRIPTION (provided by applicant): Major neurological and psychiatric diseases have been reported to be associated with significantly changed glia densities, but considerable confusion surrounds the question of the true numbers and ratios of neurons and glial cells in the brains of humans and animals. Traditionally and per "textbook knowledge," it was thought that glial cells outnumber neurons by a ratio of between 10:1 and 50:1. Recently introduced new counting techniques have challenged this belief and postulate a ratio of approximately 1:1, reducing the number of glial cells in the human brain from previous estimates by as much as 4.9 trillion (from 5 trillion to a mere 100 billion). The new, much lower estimates of glial cells are primarily based on a novel methodological approach, the isotropic fractionator (IF). However, this technique has not been calibrated or validated. It is therefore uncertain whether it produces true numbers or may possibly be biased towards larger particles (neuronal nuclei) and may underestimate smaller ones (glial nuclei). The fractionator technique homogenizes whole brains (or dissected parts thereof), then takes samples of the cell nuclei in solution, and uses antibody labeling to distinguish neuronal nuclei from other nuclei such as glia in sample solutions. While conceptually elegant, it remains to be calibrated against known numbers and ratios of neurons and glial cells. Therefore, we propose a series of calibrations that will unambiguously determine the presence or absence of any biases in the isotropic fractionator (IF) technique. We will probe a variety of human and non-human primate brain regions (primarily white matter tracts) to determine whether the fractionator accurately estimates numbers of glial cells. The same brain regions will be examined by measuring DNA content (which can directly reflect cell numbers), and by histology, using systematic random sampling to independently estimate glial numbers and ratios. Preliminary data indicate that the histological approach is most suitable to provide a gold standard for comparison. Modern stereological sampling techniques will identify the ratios of cell types at the ultrastructural level. This work will clarify true neuron-glia ratios in the bain and other parts of the CNS. Validation of the new fractionator method will be crucial for understanding neurological and psychiatric diseases with reported imbalances of glia cell densities, such as schizophrenia, autism, bipolar disease, and depression.

描述（由申请人提供）：据报道，主要的神经和精神病疾病与神经胶质密度发生了显着变化有关，但相当大的混乱围绕着人类和动物大脑中神经元和神经元细胞的真实数量和比率的问题。传统上和按“教科书知识”，人们认为神经胶质细胞的神经元比10：1至50：1的比例超过了神经元。最近引入的新计数技术挑战了这种信念，并假定比率约为1：1，将人脑中的神经胶质细胞数量从以前的估计中减少多达4.9万亿多达4.9万亿（从5万亿美元到1000亿）。新的，较低的神经胶质细胞估计值主要基于一种新型的方法论方法，即各向同性分级器（如果）。但是，该技术尚未校准或验证。因此，不确定它是否会产生真实数量或可能偏向较大的颗粒（神经元核），并且可能低估了较小的颗粒（神经胶质核）。分级器技术均质化整个大脑（或其解剖部分），然后在溶液中采集样品，并使用抗体标记将神经元核与样品溶液中的神经胶质（例如神经胶质）区分开。虽然在概念上优雅，但仍有待校准的神经元和神经胶质细胞的比例和比率均有待校准。因此，我们提出了一系列校准，这些校准将明确确定各向同性分级器（如果）技术中的任何偏见。我们将探测各种人类和非人类灵长类动物大脑区域（主要是白质区），以确定分级器是否准确估计神经胶质细胞的数量。将通过测量DNA含量（可以直接反映细胞数）和组织学来检查相同的大脑区域，并使用系统的随机抽样来独立估计神经胶质数和比率。初步数据表明，组织学方法最适合提供比较的黄金标准。现代的立体抽样技术将确定超微结构级别的细胞类型的比率。这项工作将阐明中枢神经系统的贝恩和其他部分中的真正神经元-GLIA比率。新分级方法的验证对于了解神经系统和精神病性疾病至关重要，报告的神经胶质细胞密度不平衡，例如精神分裂症，自闭症，双极性疾病和抑郁症。