1. Comparison of Plaque Burden in Tg2576 Mice Using the Aperio and TurboScan Imaging Platforms A.J. Milici

2. Alzheimer’s Disease  Alzheimer’s 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

3. Amyloid Plaque  Amyloid plaque occurs extracellular to the neuron cell body and is diagnostic of Alzheimer’s 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

4. 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

5. 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

6. 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.

7. 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.

8. 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 <180 Mb.

9. Invert contrast to enhance discrimination of plaque

10. Draw AOI around Cortex

11. Threshold and measure objects > 25 um 2

12. Objects in Hippocampus

13. Excel Data Output

14. 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%)

15. Comparison of Vehicle and MRK-560 Treatment Vehicle Treated MRK-560 Treated

16. 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.

17. 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

18. 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

19. Analysis of Aperio Scanned Sections  Images were collected at 20x  Manually draw ROIs around structures to be included or excluded from analysis

20. 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

21. Full magnification view

22. 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?

23. What may have impacted the correlation?  Artifacts Edge effect Some folds  Small “plaques” This was a 20x scan vs 10x for turbo

24. 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.

25. Acknowledgements PfizerAperio Tom KawabeSteve Potts Barbara Tate Tom Lanz Diane Stephenson