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

描述(申请人提供)：据报道，主要的神经和精神疾病与胶质细胞密度的显著变化有关，但人们对人类和动物大脑中神经元和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，根据“教科书知识”，人们认为胶质细胞的数量超过神经元的比例在10：1到50：1之间。最近引入的新计数技术挑战了这一信念，并假设了大约1：1的比例，使人类大脑中神经胶质细胞的数量从先前的估计减少了4.9万亿(从5万亿减少到仅仅1000亿)。对胶质细胞的新的、低得多的估计主要基于一种新的方法学方法，即各向同性分馏器(IF)。然而，这项技术还没有得到校准或验证。因此，不确定它是产生真实的数字，还是可能偏向较大的粒子(神经元核)，并可能低估较小的粒子(神经胶质核)。分割机技术将整个大脑(或其解剖部分)均质，然后提取溶液中的细胞核样本，并使用抗体标记来区分样本溶液中的神经元核和其他核，如胶质细胞。虽然在概念上是优雅的，但它仍然需要根据神经元和神经胶质细胞的已知数量和比例进行校准。因此，我们提出了一系列校准，明确地确定各向同性分馏塔(IF)技术中是否存在任何偏差。我们将探索人类和非人类灵长类动物大脑的各种区域(主要是白质束)，以确定分级器是否准确地估计神经胶质细胞的数量。同样的大脑区域将通过测量DNA含量(可以直接反映细胞数量)进行检查，并通过组织学进行检查，使用系统的随机抽样独立估计神经胶质细胞的数量和比率。初步数据表明，组织学方法最适合提供比较的黄金标准。现代体视学取样技术将在超微结构水平上确定细胞类型的比率。这项工作将澄清贝恩和中枢神经其他部分的真实神经元-胶质细胞比率。新分割器方法的验证对于了解神经和精神疾病，如精神分裂症、自闭症、双相情感障碍和抑郁症等已报道的胶质细胞密度失衡至关重要。

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.