Text of Comparison of Plaque Burden in Tg2576 Mice Using the Aperio and TurboScan Imaging Platforms A.J....
Comparison of Plaque Burden in Tg2576 Mice Using the Aperio and TurboScan Imaging Platforms A.J. Milici
Alzheimers Disease Alzheimers Disease (AD) is a progressive neurodegenerative disorder of the brain and is the most common form of dementia. Major contributing factor for increased risk is old age. After 65 a doubling of risk occurs every 5 years effecting ~30% of people >80 years old. Two histopathological changes within the brain. Neurofibrillary Tangles Amyloid Plaques
Amyloid Plaque Amyloid plaque occurs extracellular to the neuron cell body and is diagnostic of Alzheimers disease (AD). Plaque made up of a 42 amino acid amyloid peptide (A 42). A 42 originates from abnormal processing of Amyloid Precursor Protein (APP). APP is a transmembrane cell surface protein with functional roles in: Neurite outgrowth Cell adhesion Synaptic functions Induction of apoptosis
What has been previously done to quantify Amyloid? Many previous studies have sampled a few fields from cortex and hippocampus Issues o Variability of transgene expression o Small N o Selective field sampling bias
Methods Hemibrains were fixed ON in NBF Routinely processed and embedded in Paraffin 15 m serial sections were collected Sections were routinely stained with 6E10 antibody Images were captured: @10x using TurboScan on a Nikon--- microscope @20x using an AperioXT system TurboScan images were analyzed using ImagePro Aperio images were analyzed using the Positive Pixel Algorithm
Can we easily capture and analyze whole brain sections? Chose to analyze sagittal sections Allowed comparison of histological data to biochemical data from the same animal for both cortex and hippocampus First key question was how many sections/animal need to be analyzed Initially requested to serially section and stain an entire brain.
How many sections do we need to analyze? Able to demonstrate that 2-3 sections (15 m thick) evenly spaced resulted in a similar dataset obtained with 8 sections. This correlation held up with animals as they aged and developed more plaque.
Collect image montage @10x using TurboScan Sagittal section stained with 6E10 Mosaic made up of 60-70 images ~260 Mb File size creates ImagePro issues Requires additional pre-image processing steps to reduce image size to
Threshold and measure objects > 25 um 2
Objects in Hippocampus
Excel Data Output
Comparison of Mean % Plaque Area of Vehicle and MRK- 560 Treatment Observed a 75- 78% reduction in area of plaque. Comparable to 80% reduction in A as measured by ELISA. (-78%) (-75%)
Comparison of Vehicle and MRK-560 Treatment Vehicle Treated MRK-560 Treated
Typical Study 20 animals per group 500 sections cut per group4 days/group 60 sections stained3 days/3 groups ~15 min/slide to set up and capture3 days/group ~15 min/slide to analyze with ImagePro3 days/group Thus ~2-2.5 months to complete a typical 3-4 group study.
Why consider Aperio? Need to increase the turnaround of data Need to increase the number of groups/study Ability to easily share digital images and analysis
How are we comparing these systems? We are taking a subset of slides from an existing study These slides contained sections that varied in: o Staining intensity o Section quality o Some sections contained multiple folds Sections were analyzed using the Positive-Pixel Algorithm o Identified a set of conditions that was used on all sections
Analysis of Aperio Scanned Sections Images were collected at 20x Manually draw ROIs around structures to be included or excluded from analysis
Analyze with Positive Pixel Algorithm RegionLength (um)Positivity = NPositive/NTotal 257470.0144712 1192370.0243266 This algorithm rapidly analyzed these two ROIs Data for each ROI exported to Excel and the % positive is shown below
Full magnification view
How well does the data correlate? Very good correlation between methods in the hippocampus Correlation was OK but was not as strong in the cortex Why?
What may have impacted the correlation? Artifacts Edge effect Some folds Small plaques This was a 20x scan vs 10x for turbo
Typical study using Aperio 20 animals per group 500 sections cut per group4 days/group 60 sections stained/group3 days/3 groups ~ 2 min/slide to set up and capture Captured ON ~ 3 min/slide to analyze 2 days for entire study Using the Aperio system it will only take ~1 month to complete the study vs 2-2.5 months using our current capture and analysis methods.
Acknowledgements PfizerAperio Tom KawabeSteve Potts Barbara Tate Tom Lanz Diane Stephenson