2.1. Human subjects and tissue processing
Postmortem brain materials were collected through the Banner Sun Health Research Institute Brain and Body Donation Program (Sun City, AZ). Blocks of fixed, paraffin-embedded hippocampal tissue (at the level of the lateral geniculate nucleus) from 10 AD cases and 10 age-matched controls were obtained from the donated brains. All tissue samples were collected under institutional review board—approved protocols and informed consents permitting use of samples for research purposes. Antemortem evaluation by board-certified neurologists and neuropsychologists and postmortem evaluation by a board-certified neuropathologist were performed for all cases as described previously (Beach et al., 2012
). The diagnostic criteria followed consensus guidelines for National Institute on Aging Alzheimer’s Disease Centers. Age, sex, and postmortem interval were well matched between the groups (). The hippocampal tissue was sliced axially into 1-cm thick blocks, immersed to 4% parafolmaldehyde fixative solution for 48 hours at 4 °C and embedded in paraffin. Subsequently, the paraffin blocks were cut into 6-μm thick slices on a rotary microtome (Leica, Wetzlar, Germany), placed on microscope slides (VWR, Batavia, IL) and stored in a dark container at room temperature until further use.
Characteristics of Alzheimer's disease casees and non-demented controls
The tissue from the monozygotic twin pair was obtained from the Boston University Alzheimer’s Disease Center. Written informed consent for autopsy was obtained for both cases in compliance with institutional guidelines of Boston University. Details regarding this monozygotic twin pair discordant for AD have been described previously (Mastroeni et al., 2009
). In brief, antemortem and postmortem evaluations were performed by board-certified neurologists and a neuropathologist who determined the diagnosis according to the standard NIH AD centers protocols. The AD twin died at 76 years of age, and the non-demented control (ND) twin at 79 years of age. Postmortem delay was 7.3 hours for the AD twin and 3.1 for the ND twin. They were autopsied at the same facility using the same tissue processing protocols. Because of limitations in hippocampal tissue availability, only sections containing the CA1 region were available (protocols described below). In brief, the sections were sliced axially into 1-cm thick slabs, immersion fixed for 48 hours in 4% parafolmaldehyde at 4 °C, washed in phosphate buffer (PB), and cryoprotected in ethylene glycol and glycerol. The slabs were then sectioned at 40 μm on a cryostat. Free-floating sections were stored in freezing solution (glycol/glycerol/PB) at −20 °C until further use (Mastroeni et al., 2009
2.2. Immunohistochemistry and image analysis
For each marker, the sections were immunoreacted simultaneously using standard immunohistochemical procedures. In case of paraffin-embedded sections, deparaffination in 2 series of xylene, and rehydration in series of 100%, 96%, and 80% ethanol, and distilled water was performed before the immunohistochemical procedure. Briefly, the sections were further rinsed in phospate-buffered saline Tween-20 (PBS-T), followed by antigen retrieval using 10 mmol/L citrate buffer at 95 °C for 20 minutes, after which the sections were rinsed again with PBS-T and incubated in 1% H2O2 for 30 minutes to quench endogenous peroxidase. Subsequently, the sections were rinsed in PBS-T blocked in 3% bovine serum albumin and incubated with the primary antibodies (5-mC, diluted 1:1000, Genway Biotech, San Diego CA; 5-hmC, diluted 1:5000, Active Motif, Carlsbad, CA) overnight at room temperature. After primary antibody incubation the sections were washed again and incubated for 2 hours with the secondary antibody (goat anti-mouse alexa 488 for 5-mC and goat anti-rabbit alexa 488 for 5-hmC, 1:5000; Invitrogen, Grand Island, NY). After washing in PBS-T, the sections were counterstained with Neurotrace red fluorescent stain (Invitrogen), washed again in PBS-T, taken through Sudan Black B (Sigma Aldrich, St Louis, MO) to reduce autofluorescence, air dried overnight, and coverslipped using Vectashield mounting medium (Vector Labs, Burlingame, CA). The Neurotrace counterstain was performed so as to use neuron-specific analyses based on the characteristic morphology and size of neuronal cells when stained with this counterstain. The specificity of the 5-mC and 5-hmC primary antibodies was confirmed by using methylated or hydroxymethylated nucleotides (10 nmol/L 5-mC dCTP, 10 nmol/L 5-hmC dCTP; Zymoresearch, Irvine, CA), which brought IR levels to background signal when pre-incubated with the primary antibodies (data not shown).
An Olympus IX51 microscope (Olympus, Tokyo, Japan) was used for all imaging procedures. Images were taken with the ×40 objective using a green fluorescence filter for each staining, whereas their corresponding Neurotrace-counterstained images were taken using a red fluorescence filter. All images were taken using identical camera, microscope lens, and light settings. One hippocampal section was used per each staining, where images were taken from 5 sites in the DG, 5 sites in the CA3, and 5 sites in the CA1 per subject (total of 15 images per subject for each staining concomitant with 15 corresponding Neutrorace-counterstained images).
Fluorescence intensity analysis was performed using ImageJ software (ImageJ, U.S. National Institutes of Health, Bethesda, MD; imagej.nih.gov/ij/
). The intensity measurements were corrected for background differences by dividing the measured intensities with the average intensity of a cell-free region, such as the hippocampal white matter, in each section. For each image, the fluorescence intensity of 15 individual neurons and 15 individual cells of non-neuronal origin were analyzed by delineating the nucleus of each cell and measuring the mean intensity value. Thus, the fluorescence intensity of a total of 450 cells per individual was analyzed per staining. The discrimination between cells of neuronal and non-neuronal origin was done based on their characteristic morphology and size of neurons when using the Neurotrace counterstain (Sekirnjak et al., 2003
). According to these criteria, a clear differentiation between neurons and glia in the DG was not possible, and thus no cell-type specific analyses were carried on in this subregion. Of note, the measurements of fluorescence intensities were calculated in arbitrary units and do not represent absolute levels of these markers ().
Fig. 1 Hippocampal 5-methylcytidine (5-mC) immunoreactivity (IR) and Neurotrace counterstain. Representative photomicrograph of 5-mC IR (green) and the corresponding Neurotrace image (red) taken from the CA1 hippocampal subregion. Based on the Neurotrace morphology, (more ...)
2.3. Statistical analysis
A multilevel linear mixed model was used for the statistical analyses. For each marker, the fluorescence intensities were used as the dependent variable and the AD diagnosis as the independent variable. Measurements of individual cells (level 1) were clustered in the different subjects (level 2). Statistical significance was set at α = 0.05. Following the multilevel analyses, stratified analyses for neuronal cells and cells of non-neuronal origin (glial cells) were performed for each hippocampal subregion (CA3, CA1-2) or just for neuronal cells (DG) separately using the same statistical model. Correlation analyses were carried out by calculating the Pearson’s correlation coefficient (rp). All statistical calculations were performed using STATA 11 (StataCorp, College Station, TX) or the Statistical Package for the Social Sciences (SPSS 17; SPSS Inc, Chicago, IL). Graphs were built in GraphPad Prism (Version 4, GraphPad Software, San Diego, CA).