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

描述（由申请人提供）：据报道，主要的神经和精神疾病与神经胶质密度的显著变化有关，但围绕人类和动物大脑中神经元和神经胶质细胞的真实数量和比例的问题存在相当大的困惑。传统上和根据“教科书知识”，人们认为神经胶质细胞的数量超过神经元的比例在10：1和50：1之间。最近引入的新计数技术挑战了这一信念，并假设比例约为1：1，将人脑中神经胶质细胞的数量从之前的估计减少了4.9万亿（从5万亿减少到仅仅1000亿）。新的，低得多的估计神经胶质细胞主要是基于一种新的方法，各向同性分馏器（IF）。然而，这项技术尚未经过校准或验证。因此，不确定它是否产生真实的数字，或者可能偏向于较大的颗粒（神经元细胞核），并可能低估较小的颗粒（神经胶质细胞核）。分馏器技术将整个大脑（或其解剖部分）均质化，然后在溶液中采集细胞核样品，并使用抗体标记来区分神经元核与样品溶液中的其他核（如神经胶质）。虽然在概念上很优雅，但它仍然需要根据已知的神经元和神经胶质细胞的数量和比例进行校准。因此，我们提出了一系列的校准，将明确地确定存在或不存在任何偏见的各向同性分馏器（IF）技术。我们将探测各种人类和非人类灵长类动物的大脑区域（主要是白色物质束），以确定是否分馏准确估计神经胶质细胞的数量。将通过测量DNA含量（可直接反映细胞数量）和组织学检查相同的脑区域，使用系统随机抽样独立估计神经胶质细胞数量和比例。初步数据表明，组织学方法是最适合提供比较的金标准。现代体视学取样技术将在超微结构水平上确定细胞类型的比例。这项工作将澄清真正的神经元胶质细胞比例在贝恩和其他部分的中枢神经系统。新的分馏器方法的验证将是至关重要的，以了解神经和精神疾病与报告的胶质细胞密度的不平衡，如精神分裂症，自闭症，双相情感障碍和抑郁症。

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.