The reliability of the isotropic fractionator method for counting total cells and neurons

Abstract

Background:The Isotropic Fractionator (IF) is a method used to determine the cellular composition of nervous tissue. It has been mostly applied to assess variation across species, where differences are expected to be large enough not to be masked by methodological error. However, understanding the sources of variation in the method is important if the goal is to detect smaller differences, for example, in same-species comparisons. Comparisons between different mice strains suggest that the IF is consistent enough to detected these differences. Nevertheless, the internal validity of the method has not yet been examined directly. Method:In this study, we evaluate the reliability of the IF method for the determination of cellular and neuronal numbers. We performed repeated cell counts of the same material by different experimenters to quantify different sources of variation.Results: In total cell counts, we observed that for the cerebral cortex most of the variance was at the counter level. For the cerebellum, most of the variance is attributed to the sample itself. As for neurons, random error along with the immunological staining correspond to most of the variation, both in the cerebral cortex and in the cerebellum. Test-retest reliability coefficients were relatively high, especially for cell counts. Conclusions:Although biases between counters and random variation in staining could be problematic when aggregating data from different sources, we offer practical suggestions to improve the reliability of the method. While small, this study is a most needed step towards more precise measurement of the brain's cellular composition.

The number of neurons in Drosophila and mosquito brains

“…Stereological counting is applied to heterogeneous brain structures by subdividing the structure into smaller, more uniform components. However, this approach is prone to error because estimates are obtained from cell densities and based on the homogeneity of nuclei in the sample [10][11][12]. Minimizing this error would require the burden of subdividing the brain into smaller parts with homogenous cell density.…”

“…The isotropic fractionator (IF) method involves the combination of direct enumeration and immunohistochemistry, and has the advantage of circumventing the drawbacks in stereological methods [10][11][12]. The IF method involves the conversion of non-uniform tissue structure into a homogenous single-cell suspension [13] (Fig 1).…”

Effects of nasal parasite species in the small-spotted catshark Scyliorhinus canicula (Scyliorhinidae; Carcharhiniformes)

“…The evaluation of the number of cells in the parasitized OB showed a lower number of neurons and nonneuron cells compared to the contralateral OB, and also to the average OB of unparasitized S. canicula of a similar size range (Table 2). The differences are noteworthy and unlikely could be due to error associated with the isotropic fractionator technique (Neves et al 2019, Aicardi et al 2020). If the copepod had hindered the shark's olfaction, the effect would be comparable to olfactory deprivation and it could be the cause of the neuron number decrease in the OB, like that seen in mammals and bony fishes.…”

Alteration in the number of neuronal and non-neuronal cells in mouse models of obesity

“… 20 , 51 Moreover, the IF method has better staining with NeuN antibodies and eliminates problems with tissue shrinkage, which leads to more reliable results when analysing neuronal populations. 51 , 52 Therefore, IF has the potential to more precisely evaluate the cellular composition of specific brain regions in obese mouse models.…”