high-throughput3d whole-brain quantitative histopatology
TRANSCRIPT
HIGH-THROUGHPUT 3D WHOLE-BRAIN
QUANTITATIVE HISTOPATOLOGY
IN RODENTS
GdR – ISIS : Analyse de tissu biologique
et histopathologie numérique
Michel Vandenberghe, Anne-Sophie Hérard,
Zhenzhen You, Nicolas Souedet, Philippe Hantraye,
Marc Dhenain, Thierry Delzescaux
CEA-MIRCen Molecular Imaging Research Centre
UMR 9199, CNRS/CEA
Laboratoire des Maladies Neurodégénératives
Molecular Imaging Research Centre - CEA
Therapies
Mechanisms
Imaging
An integrated platform:
– Housing and experimentation A1/A2/A3
– MRI – MRS
– PET
– Histology
– Microscopy
– Image analysis
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ParkinsonAlzheimer
Huntington
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Resolution (m)
110-110-210-310-410-510-610-710-810-9
PET
MRI
Optical microscopy
Electron microscopy
Histopathology in Alzheimer’s Disease (AD)
3Holtzman et al, 2011
• High-specificity
• High-resolution
• Post mortem
• Two-dimensional
Amyloid IHC Tau IHC
+
-
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Literature survey: amyloid load quantification
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0
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120
1999200020012002200320042005200620072008200920102011
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ber
of p
ublic
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ns
Years
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
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ber
of p
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Number of sections analyzed per animal
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1 2 3 4 5 6 7
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ber
of p
ublic
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Number of ROIs
Publications with amyloid load quantification How ma ny sections ?
How many ROIs ? How are amyloid plaques segmented ?
→ Cerebral cortex & hippocampus
0 5 10 15 20
Number of publications
Seg
men
tatio
n m
etho
d
Manual
Stereology
Not specified
Threshold
Interest to extend the analysis to 3D ?
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3D whole-brain histopathology: amyloid load
Applications of brain-wide quantitative histopathology at the mesoscopic scale:
- Mouse model characterization
- Preclinical evaluation of drug candidates
- Gene effect on amyloidosis
- In vivo imaging validation
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Resolution (m)
110-110-210-310-410-510-610-710-810-9
PET
MRI
Optical microscopy
Electron microscopy
3D whole-brain histopathology
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Material and methods
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Experimental designs
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• First study:– 13.5 months old APP/PS1dE9 mice (n=6)
→ Model characterization
• Second study:– 12 APP/PS1 mice:
• 8 were treated with 13C3a
• 4 were treated with DM4 (treatment control group)
– 4 PS1 mice (amyloid plaque free mice).
– Mice were imaged by MRI for amyloid plaques before euthanasia
(Marc Dhenain, Mathieu Santin).
→ Method validation and comparison with classical quantification approach
→ 13C3a immunotherapy evaluation
→ Spa<al correlation of pathological and cellular markers
→ in vivo - post mortem confrontation
• Third study:APPswe mice (n=5), APPswe-ADAM30 mice (n=5),
APPswe-ADAM30-Cre (n=10), other groups…
→ What is the effect of ADAM30 expression on amyloid load?
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Histology processing
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• Block-face photographs were taken during brain
cutting (lateral resolution: 33 µm)
• Sections were stained for :
– Anatomy (Nissl staining)
– Aβ plaques (6E10 IHC)
– Mouse IgG1 (GAM IHC)
– Microglia (Iba-1 IHC)
– Activated microglia (CD-68 IHC)
• Images are acquired at 21 µm/pixel for Nissl series
and at 5 µm/pixel for the other series.
6E10Nissl IgG1 Iba-1 CD-68
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Section to sectionco-alignement
Spatial reference
Image stacking
Series 1 Series n
Histological imagesBlock-face
photographs…
Histological images stacks
3D reconstruction
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Cresyl violet volume6E10 IHC volume IgG IHC
volumeBlock-face photographs
volume (spatial reference)
GdR ISIS, 23 juin 2015 - T. Delzescaux
Section to sectionco-alignement
Spatial reference
Image stacking
Series 1 Series n
Histological imagesBlock-face
photographsMouse brain
atlas
Linear andnon-linear
registration
…
Histological images stacks
Atlas based analysis (1/3)
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Cresyl violet volume6E10 IHC volume IgG IHC
volumeBlock-face photographs
volume (spatial reference)Mouse brain altas
http://www.mouseimaging.ca/technologies/
C57Bl6j_mouse_atlas.html
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Quantification of histological markers
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→ Histological volumes are segmented
→ Segmented volumes are in the spatial reference
→ Data output compa<ble with statistical
software (Statistica, R…)
6E10 IHC
Anti-IgG IHC
Section to sectionalignement
Spatial reference
Image stacking
Series 1 Series n
Automatic segmentation
1110010011010
11001
1110010011010
11001
Segmented histology volumes
Histology imagesBlock-face
photographsMouse brain
atlas
Data output
Linear andnon-linear
registration
…
Histology image stacks
Atlas based quantification
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6E10 IHC segmented volume
IgG IHC segmentedvolume
Mouse brain altas
Applications
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Mouse model characterization
→ Amyloid load deposition +++ in cerebral cortex and hippocampal region
→ Amyloid deposition detected in basal ganglia and white matter tracts
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Immunotherapy evaluation
→ Amyloid load is close to none in PS1 mice
→ 13C3a lowered amyloid load in APP/PS1 mice.
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Vandenberghe et al.,
in revision.
ADAM30 overexpression effect on amyloid load
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Letronne et al., in preparation
Vandenberghe et al., in preparation.Atlas-based analysis
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Validation and comparison
with
current standards
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Profiling amyloid load distribution along the rostro-caudal
axis
ROIMean coefficient of
variation (n=11)
Cerebral cortex 0.39
Hippocampal region 0.44
Striatum 0.38
Thalamus 0.55
Amyloid load variation along the rostro-caudal axis
(n=12 APP/PS1 mice)Mouse brain ROIs in 3D
Validation and comparison with current standards
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Perspectives and
preliminary results
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Perspective: microscopic image analysis
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ImagesResolution
(xyz, µm)
Approximate
number of
voxels
Approximate
file size
(gygabytes)
7T mouse brain MRI scan 30 30 30 107 0.02
Block-face photography
volume
(100 sections)
30 30 120 107 0.02
Mesoscopic IHC volume
(100 sections)5 5 120 108 1
Microscopic IHC volume
(100 sections)0.20 0.20 120 1011 500
Resolution (m)
110-110-210-310-410-510-610-710-810-9
Zeiss Axio Scan.Z1
Brain-wide mesoscale histology
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Multiple markers 3D histopathology [1/3]
Information
acquired at
cellular level
Analysis
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From mesoscopic to microscopic scale (1/4)
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Multiple markers 3D histopathology [2/3]
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From mesoscopic to microscopic scale (2/4)
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From mesoscopic to microscopic scale (3/4)
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From mesoscopic to microscopic scale (4/4)
(Zhenzhen YOU)
Mouse brain section (Iba1)
Microglia cell
New challenges to adress: cell indidualization, big data processing, etc...
GdR ISIS, 23 juin 2015 - T. Delzescaux
Direction des Sciences du Vivant
I2BM
MIRCen
Commissariat à l’énergie atomique et aux énergies alternatives
Centre de Fontenay-Aux-Roses | 92265 Fontenay-Aux-Roses Cedex
T. +33 (0)1 46 54 84 11 | [email protected]
Etablissement public à caractère industriel et commercial | RCS Paris B 775 685 019
Marc Dhenain
Caroline Jean
Fanny Petit
Mathieu Santin
Didier Thenadey
Kelly Herbert
Thierry Delzescaux
Anne-Sophie Hérard
Nicolas Souedet
Elmahdi Sadouni
Yaël Balbastre
Zhenzhen You
Frédérique Frouin
Laurent Pradier
Tom Rooney
Thomas Debeir
Véronique Blanchard
Dominique Briet
Pierre Etienne Chabrier
Michel Auguet
Sylvie Cornet
Brigitte Spinnewyn
Denis Carré
Jocelyne Schultz
Bob Switzer
Robert Switzer
Programme Technologie Santé – Programme Irtelis
Jean-Charles Lambert
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