Abstract
It is of critical importance to understand the numbers and distributions of neurons and non-neurons in the cerebral cortex because cell numbers are reduced with normal aging and by diseases of the CNS. The isotropic fractionator method provides a faster way of estimating numbers of total cells and neurons in whole brains and dissected brain parts. Several comparative studies have illustrated the accuracy and utility of the isotropic fractionator method, yet it is a relatively new methodology, and there is opportunity to adjust procedures to optimize its efficiency and minimize error. In the present study, we use 142 samples from a dissected baboon cortical hemisphere to evaluate if isotropic fractionator counts using a Neubauer counting chamber and fluorescence microscopy could be accurately reproduced using flow cytometry methods. We find greater repeatability in flow cytometry counts, and no evidence of constant or proportional bias when comparing microscopy to flow cytometry counts. We conclude that cell number estimation using a flow cytometer is more efficient and more precise than comparable counts using a Neubauer chamber on a fluorescence microscope. This method for higher throughput, precise estimation of cell numbers has the potential to rapidly advance research in post-mortem human brains and vastly improve our understanding of cortical and subcortical structures in normal, injured, aged, and diseased brains.
Highlights
In the cerebral cortex changes in numbers of neurons accompany normal aging as well as a variety of neurological disorders including Alzheimer’s disease and Parkinson’s disease and developmental disorders such as autism
We estimated numbers of neurons and non-neurons across the cortical sheet in a baboon cortical hemisphere using the isotropic fractionator method combined with flow cytometry, and compared those results with the isotropic fractionator method and microscope counts using a Neubauer chamber
We expect error produced by processing, if any, to be the same or very similar between microscope-counted samples and flow cytometry-counted samples because all samples were treated identically, with the exception of using a goat anti-mouse secondary antibody tagged with a different Alexa Fluor color [AF 594 vs.AF 700]
Summary
In the cerebral cortex changes in numbers of neurons accompany normal aging as well as a variety of neurological disorders including Alzheimer’s disease and Parkinson’s disease and developmental disorders such as autism. Methods for counting cells have been tailored toward evaluating cell numbers on a small scale in well-defined structures in the brain. With the recent development of the isotropic fractionator method (HerculanoHouzel and Lent, 2005), it has become possible to accurately and rapidly evaluate a larger number of samples from a wider variety of structures, provided they are accurately dissectable. The isotropic fractionator method (Herculano-Houzel and Lent, 2005) provides a faster way of estimating numbers of total cells and neurons of whole brains and dissected brain parts. The isotropic fractionator circumvents the complexity of summing multiple, small, more uniform samples of brain structures by converting any heterogeneous structure into a homogeneous one by completely dissociating the cells into a uniform nuclear suspension. The speed and practicality of the isotropic fractionator make our studies achievable in a relatively short span of time
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