Validation of the isotropic fractionator: comparison with unbiased stereology and DNA extraction for quantification of glial cells

Abstract

Background: The "isotropic fractionator" (IF) is a novel cell counting technique that homogenizes fixed tissue, recovers cell nuclei in solution, and samples and quantifies nuclei by extrapolation. Studies using this technique indicate that the ratio of glia to neurons in the human brain is approximately 1:1 rather than the 10:1 or 50:1 ratio previously assumed. Although some results obtained with the IF have been similar to those obtained by stereology, the IF has never been calibrated or validated. It is conceivable that only a fraction of glial cell nuclei are recovered intact or recognized after the homogenization step.

New method: To rule out this simple explanation for the claim of a 1:1 glia-neuron ratio, we compared cell numbers obtained from adjacent, weight-normalized samples of human and macaque monkey white matter using three techniques: the IF, unbiased stereology of histological sections in exhaustively sectioned samples, and cell numbers calculated from DNA extraction.

Results and comparison of methods: In primate forebrains, the IF yielded 73,000-90,000 nuclei/mg white matter, unbiased stereology yielded 75,000-92,000 nuclei/mg, with coefficients of error ranging from 0.013 to 0.063, while DNA extraction yielded only 4000-23,000 nuclei/mg in fixed white matter tissues.

Conclusions: Since the IF revealed about 100% of the numbers produced by unbiased stereology, there is no significant underestimate of glial cells. This confirms the notion that the human brain overall contains glial cells and neurons with a ratio of about 1:1 - far from the originally assumed ratio of 10:1 in favor of glial cells.

Keywords: Bias; Brain; Calibration; Glia-neuron ratio; Glial cell; Human; Isotropic fractionator; Primate; Quantification; Stereology; White matter.

Fig. 1

A–B. Examples of formaldehyde-fixed human and monkey hemi-brains showing the locations where adjacent samples were taken. Samples were obtained in the corpus callosum (red squares) and in the cerebellum (red asterisks) for subsequent processing by either the isotropic fractionator, by stereology, or (forebrain only) by DNA extraction. A. Human brain. B. Cynomolgus macaque brain. Scale bars = 1 cm.

Fig. 2

A–E. Examples of stained cell nuclei measured with the isotropic fractionator (IF) in solution or in histological sections. A. Cell nuclei isolated from human white matter, stained with the fluorescent nuclear stain DAPI, and counted in a hemocytometer. B. Histological section of macaque white matter containing cell nuclei stained with DAPI. An unbiased counting box is shown, as used for counting of stained nuclei. Scale bar = 10 μm. C. Histological section of macaque white matter containing glial cell nuclei stained with thionin. An unbiased counting box is shown, as used for counting of stained nuclei. Scale bar = 10 μm. D. The sizes of cell nuclei from human white matter (red bars) show a normal distribution with a peak at 50 μm² when plotted for frequency in IF analysis. Cell nuclei from grey matter (blue bars) show multiple, broader peaks of maximal nuclear area, consistent with additional peaks of smaller and larger neuronal nuclei at 60 and 130 μm². E. The z-axis distribution of thionin-stained cell nuclei in white matter from macaque brain. The top of the tissue section (exposed to the cover slip) is shown as the 10 percentile, the bottom of the tissue section (against the glass slide) as the 100 percentile. Note the drop-off at the bin closest to the section surfaces, indicative of lost caps, and the two peaks at the 30^th and 70–80^th percentiles, indicative of section compression. The number (n) of sampled nuclei is indicated.

Fig. 3

A–E. Comparison of cell numbers obtained by three different quantification techniques, the isotropic fractionator (IF), stereology of histological sections (Histo), and DNA extraction. All error bars=SEM. A. Comparison of IF, stereology and DNA-based quantification of cells in human and macaque monkey forebrain. emb, embalmic fixation; for, formalin fixation; ca, callosum; co, cortex. Note that IF and stereology yield about 80,000–90,000 cells/mg, while DNA extraction yields only a fraction (20,000 cells/mg). B. A paired Student’s t-test shows no statistically significant (n.s.) difference between the IF and stereology (p>0.4; the number on the bars indicates the number of independent experiments); data shown are for IF and stereology for formalin-fixed human forebrain samples and paraformaldehyde-fixed monkey forebrain samples. The number on each bar indicates the number of independent experiments. C. When stereology data (from panel B) are adjusted for maximal possible bias due to lost caps (+14%), the difference between the IF and stereology is still not significant (paired Student’s t-test). D. The glial density of human white matter differs from that of macaque white matter forebrain (Student’s t-test, using both IF and stereology data), but only when embalmic human samples are included; the difference in cerebellum was not significant; Cb, cerebellum; Fb, forebrain; hu, human; mo, monkey; n.s., not significant. E. The cerebellar white matter contains significantly lower glial cell densities than cortical white matter, in both human and macaque (based on both IF and stereology data).