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

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.