reviewed paper: Iacono, D., Resnick, S.M., O’Brien, R., Zonderman, A.B., An, Y., Pletnikova, O, Rudo, G., Crain, B., and J.C. Troncoso (2014) Mild Cognitive Impairment and Asymptomatic Alzheimer Disease Subjects: Equivalent β-Amyloid and Tau Loads With Divergent Cognitive Outcomes. J. Neuropathol. Exp. Neurol. 73(4): 295-304.
Juan Troncoso, MD and Gay Rudo have considerable expertise using unbiased stereology to study Alzheimer’s disease (AD). In this published study (Iacono, et al., 2014) the stereological method of “point-counting” is used to show that brains from subjects from the Baltimore Longitudinal Study of Aging that are considered to have had asymptomatic AD do not show more plaque-load of β-amyloid or phosphorylated tau in regions vulnerable to AD pathology than brains from subjects that had mild cognitive impairment. Plaque load, unlike hypertrophy of neurons (Iacono et al., 2008), does not correlate with asymptomatic AD. The hypertrophy of neurons is hypothesized to be indicative of a compensatory mechanism while the presence of plaque may not be a causal factor in cognitive impairment associated with AD.
The hypothesis in this study is that brains of asymptomatic AD subjects have less amounts of plaque than brains from subjects with mild cognitive impairment in CA1, anterior and posterior cingulate gyrus, and visual cortex. The experimental results in this study reject this hypothesis; for the most part, there is not less plaque by volume in the brains from asymptomatic AD subjects (Iacono, et al., 2014, Fig. 3 and 4, note that the Y-axis is labeled as a percentage). To qualify as an asymptomatic AD subject, the brain of a person without any behavioral symptoms must have AD lesions, meaning β-amyloid neuritic plaques, neurofibrillary tangles, and neuropil threads. The presence of these lesions is assessed using a semi-quantitative scoring system (Iacono, et al., 2014, Introduction). The authors wanted to use a quantitative method to look at the percent plaque by volume in an unbiased way in these experimental groups, so they chose unbiased stereology. It is always best to use systematic random sampling if possible. In this case, as it often is with human tissue, random tissue blocks were dissected from the brain regions (Iacono et al., 2008, Neuropathology Methods and Diagnostic Criteria) instead of using systematic random sampling. Sections were ten microns thick; the use of sections that are relatively thin compared to the whole structure avoids over-projection effects when performing point-counting. One slide was randomly picked from each region, again deviating from the ideal use of systematic random sampling. At low magnification (5x objective lens) the region of interest was defined; areas where the most plaque was qualitatively visible were outlined; this may introduce bias compared to a method that picks the region of interest based on anatomical borders. The area fraction fractionator probe was conducted on the regions of interest at a higher magnification (40x objective lens); the grid step or area between counting frames was 450 X 450 microns, the counting frame size is not given, and the counting frame contained points that were spaced 15 microns apart. One type of marker was placed on points over plaque and another type was placed on non-plaque tissue. The estimate of the percentage of plaque by volume is calculated as the number of plaque markers divided by the number of plaque plus non-plaque markers (Iacono, et al., 2014, Unbiased Stereological measurements).
The authors report that this study is one of the first to use unbiased stereology, namely the area fraction fractionator, to assess plaque load in brains from symptomatic vs. asymptomatic AD subjects. We encourage the use of this probe rather than tracing because of its efficiency and unbiased nature and the fact that it is used with thin sections and does not require any special manipulation of the tissue allowing the use of preferentially-oriented sections. It is laudable that most of the stereological parameters are published in the paper to allow reproduction of this experiment..
Iacono, D., O’Brien, R., Resnick, S.M., Zonderman, A.B., Pletnikova, O, Rudow, G., An, Y., West, M.J., Crain, B. and J.C. Troncoso (2008) Neuronal Hypertrophy in Asymptomatic Alzheimer Disease. J. Neuropathol. Exp. Neurol. 67(6): 578 – 589.
Iacono, D., Resnick, S.M., O’Brien, R., Zonderman, A.B., An, Y., Pletnikova, Ol, Rudo, G., Crain, B., and J.C. Troncoso (2014) Mild Cognitive Impairment and Asymptomatic Alzheimer Disease Subjects: Equivalent β-Amyloid and Tau Loads With Divergent Cognitive Outcomes. J. Neuropathol. Exp. Neurol. 73(4): 295-304