Abstract
One major aim in quantitative and translational neuroscience is to achieve a precise and fast neuronal counting method to work on high throughput scale to obtain reliable results. Here, we tested the isotropic fractionator (IF) method for evaluating neuronal and non-neuronal cell loss in different models of central nervous system (CNS) pathologies. Sprague-Dawley rats underwent: (i) ischemic brain damage; (ii) intraperitoneal injection with kainic acid (KA) to induce epileptic seizures; and (iii) monolateral striatal injection with quinolinic acid (QA) mimicking human Huntington’s disease. All specimens were processed for IF method and cell loss assessed. Hippocampus from KA-treated rats and striatum from QA-treated rats were carefully dissected using a dissection microscope and a rat brain matrix. Ischemic rat brains slices were first processed for TTC staining and then for IF. In the ischemic group the cell loss corresponded to the neuronal loss suggesting that hypoxia primarily affects neurons. Combining IF with TTC staining we could correlate the volume of lesion to the neuronal loss; by IF, we could assess that neuronal loss also occurs contralaterally to the ischemic side. In the epileptic group we observed a reduction of neuronal cells in treated rats, but also evaluated the changes in the number of non-neuronal cells in response to the hippocampal damage. In the QA model, there was a robust reduction of neuronal cells on ipsilateral striatum. This neuronal cell loss was not related to a drastic change in the total number of cells, being overcome by the increase in non-neuronal cells, thus suggesting that excitotoxic damage in the striatum strongly activates inflammation and glial proliferation. We concluded that the IF method could represent a simple and reliable quantitative technique to evaluate the effects of experimental lesions mimicking human diseases, and to consider the neuroprotective/anti-inflammatory effects of different treatments in the whole brain and also in discrete regions of interest, with the potential to investigate non-neuronal alterations. Moreover, IF could be used in addition or in substitution to classical stereological techniques or TTC staining used so far, since it is fast, precise and easily combined with complex molecular analysis.
Highlights
Since its introduction (Herculano-Houzel and Lent, 2005), the isotropic fractionator (IF) method represented a significant innovation in the field of quantitative neuroscience
We found a significant difference in the number of 4 -6diamino-2-phenylindole dihydrochloride (DAPI) positive cells in ischemic brains compared to the controls (1.06∗108 ± 3.95∗106 vs. 1.33∗108 ± 7.42∗106, p = 0.04)
The IF method was previously adopted for determining age-related neuronal loss in rats (Morterá and Herculano-Houzel, 2012), synucleinopathy (Aldrin-Kirk et al, 2014), in a mouse model of Alzheimer’s disease (AD) (Brautigam et al, 2012) and even on AD human patients (Andrade-Moraes et al, 2013)
Summary
Since its introduction (Herculano-Houzel and Lent, 2005), the isotropic fractionator (IF) method represented a significant innovation in the field of quantitative neuroscience. The issue of counting neurons and other cell types in the nervous system has been fundamental in neuroscience since the past century. Generating accurate and reproducible quantitative estimates of both neuronal and non-neuronal populations is a crucial requirement in a number of experimental setups aiming at tracking developmental or neurodegenerative events in the nervous system, yet available protocols are hampered by a number of technical and procedural limitations. A series of methods was developed in order to overcome the problem of overestimating cell number due to double counting of profiles in histological sections, because not all of the counted objects are contained exclusively in one section (Abercrombie, 1946). The bigger the nuclear dimension is, in relation to the section thickness, the bigger the error could be
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