2.2.1
Animals
All animal studies were conducted in accordance with the guidelines of the Subcommittee on Animal Care at the University of Western Ontario, and conformed to the Canadian Council on Animal Care guide for the care and use of laboratory animals. The required documentation is attached in Appendix 5. All mice were received from Jackson Laboratories (Bar Harbor, ME, USA). Three Tg mouse models of AD were selected. In the current study, a total of three female mice per time point (age) per strain were used (N=63) in total; eighteen controls, fifteen 5X (5XFAD), fifteen 2X (APPswe/PS1dE9) and fifteen 3X (3xTg) mice. All animals, which were used up to twelve months of age, were housed with a 12-h light/12-h dark photoperiod and received food and water ad libitum.
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2.2.2
Preliminary tissue processing
At each time point (age group), mice were placed into a surgical plane by induction chamber CO2 inhalation followed by severing the diaphragm, left ventricle
heparin injection (2 ml/kg), right atrium cut and ice-cold phosphate buffered saline (PBS) perfusion (at least 10 ml) via the left ventricle catheter and lastly decapitation. Brains were rapidly removed and dissected while kept on ice. One hemisphere was removed and immersed in 10% neutral buffered formalin NBF immersion for at least another 48 hours prior to additional processing. The other hemisphere (contralateral) was dissected further to separate the hippocampus, cortex, and striatum and then stored at -80°C. Tissue collection was performed at 5, 16, 32, 44 and 60 weeks of age for the 2X and 3X mice, 5, 8, 16, 28 and 60 weeks of age for the 5X mice; and finally 5, 8, 16, 32, 44, 60 weeks of age for the WT mice.
2.2.3
Western Blotting
Frozen tissue samples were combined in ice cold lysis buffer [20mM Tris–HCl (pH 8), 150 mM NaCl, 0.1% SDS, 1mM EDTA, 1% Igepal CA-630, 50mM] with protease cocktail inhibitors with pepstatin A (Roche, Mississauga, Ontario, Canada) and homogenized using a Pestle Grinder System (Thermo Fisher Scientific, Massachusetts, USA) and frozen at -80°C. Lanes were run with 20 μg of protein on a 12% SDS– polyacrylamide gel and the resolved proteins were transferred to a nitrocellulose membrane (all from Bio-Rad, Mississauga, Ontario, Canada) using a Trans-Blot Semi- Dry Electrophoretic Transfer Cell (Bio-Rad, Mississauga, Ontario, Canada). The membrane was blocked with 5% nonfat milk (w/v) dissolved in Tris buffered saline with 0.05% Tween-20 (TBST). Blots were probed using Anti-Cat-D antibody at 1:300 dilution factor (chosen to ensure a linear response across a several fold increase in the concentration of target protein) overnight (C-20- sc6486; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and detected using rabbit-anti-goat antibody coupled to horseradish peroxidase using enhanced chemiluminescence reagents (ECL;). Blots were
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imaged digitally using the Gel Doc XR+ System (BIORAD; California, USA). Blots were then washed 3 times in TBST and re-probed with anti-α-tubulin at 1:50k dilution factor overnight (Sigma Aldrich, St Louis, MO, USA) as a loading control and imaged the following day. Since immature Cat-D is also the active form and performs the same enzymatic functions as the mature form, both forms were grouped into a total CatD expression {Benes, 2008, Cathepsin D--many functions of one aspartic protease}. Images were analyzed using ImageJ (http://rsbweb.nih.gov/ij/). CatD chemiluminescent bands were evaluated relative to the loading control measurement of α-tubulin as follows using Equation (2-1):
𝑰
𝑰 𝒏 (2-1)
Where ψ is the ratio of CatD compared to α-tubulin. The SICatD or SIα_tubulin are the
densitometric “signals” measured from the blot for either CatD or α-tubulin respectively, i = 1..3 are the number of animals used. Next, in order to normalize all data across all strains, brain regions and ages - all ψ (of any strain, age, or mouse) were normalized to
the ψ of a single five week old WT mouse cortex based on the relative CatD expression
previously measured using a separate WB analysis for all 5 week old WT tissues (9 samples: 3 mice, 3 brain regions). This process was completed for each mouse, at each time point, for each brain region (striatum, hippocampus and cortex).
2.2.4
Immunohistochemistry Processing and Analysis of
Transgenic and Wild Type Mice
All brains were allowed to fix over time in 10% NBF before switching to 70% ethanol and embedded in paraffin. Five (5) µm thick sections were cut in a coronal orientation to expose the hippocampus, striatum, cortex, as well as the thalamus. All sections were stained (for each primary antibody) at the same time for standardization purposes. In addition, any potential variability in staining between the sections, animals and time points was minimized by using an automatic threshold as described in more
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detail below. Endogenous peroxidase activity was inhibited by using hydrogen peroxide (3%) followed by antigen retrieval using formic acid (70%, 2 minutes) and sections were immunolabeled with either an antibody against CatD (1:200 dilution factor; C-20 sc-6486 from Santa Cruz Biotechnology; Santa Cruz, CA, USA) or against Aβ42 (1:200 dilution
factor; custom made). The dilution factors were chosen after performing serial dilutions of different concentration. A dilution was chosen that did not produce excessive staining of CatD in the younger mice (as well as a linear response in Western blots). In addition, dilution factor was chosen for the Aβ42 antibody to produce no staining of note in the
older WT mice (non-zero values measured in WT mice set base-line for thresholding/ colour deconvolution). This antibody was detected using an anti-rabbit secondary antibody conjugated to horseradish peroxidase, prior to colorimetric detection using nickel 3,3’-diaminobenzidine (DAB; 0.15 mg/ml in 0.03% H2O2; Sigma-Aldrich,
Oakville,ON, Canada) with the Vectastain ABC kit (Dako, Burlington, ON, Canada) and counterstaining with Hematoxylin and Eosin (H&E; Leica Microsystems Inc. Concord, Ontario, Canada). Negative control (no primary antibody) did not produce any notable staining. Sections were imaged and digitized using a TISSUEscope CF slide scanner (Huron Technologies, Waterloo, ON) at a 0.5 µm resolution using bright field imaging (at 40X). Slides were measured for positively stained areas in four brain regions (each covering >200,000 µm2) per mouse, in each of three mice per age group using ImageJ (v. 1.46r, http://rsbweb.nih.gov/ij/). Staining was identified using the color deconvolution plugin written by Dr. Gabriel Landini (available via the aforementioned website) and quantified using the ImageJ built-in threshold tool (triangle thresholding with seldom minor manual adjustments if deemed necessary by the primary observer JAS). To assess intra-observer variability, a subset of the data (Aβ42) was analyzed by a second blinded
observer (blinded to strain, age, and tissue type).
2.2.5
Statistical analysis
Two-tailed, unequal variance Student’s t-test was used to test for significant differences correcting for multiple comparisons using the Sidak-Bonferroni method using Prism5 (GraphPad software, Inc. La Jolla, CA, USA) comparing the amount of DAB staining detected in the different brain regions via both Western blots and
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histopathological examination. Additionally, a two-way ANOVA with Tukey’s correction for multiple comparisons was used to examine the influence and statistical differences as a result of having AD mutations (strain factor) and aging (age factor). For ANOVA, when comparing WT mice with 2X or 3X, the missing 32-week old WT mice data assumed the values of the available 28-week old data. Finally, a Pearson correlation test as well as Student’s t-test was used to check for intra-observer variability and correlation of the measurements performed for CatD and Aβ42 staining.