HIGH-THROUGHPUT3D WHOLE-BRAIN QUANTITATIVE HISTOPATOLOGY ...

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

[email protected]

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

2

ParkinsonAlzheimer

Huntington

GdR ISIS, 23 juin 2015 - T. Delzescaux

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

+

-


Literature survey: amyloid load quantification

4

0

20

40

60

80

100

120

1999200020012002200320042005200620072008200920102011

Num

ber

of p

ublic

atio

ns

Years

0

2

4

6

8

10

12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Num

ber

of p

ublic

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ns

Number of sections analyzed per animal

0

5

10

15

20

25

1 2 3 4 5 6 7

Num

ber

of p

ublic

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ns

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 ?


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

5

Resolution (m)

110-110-210-310-410-510-610-710-810-9

PET

MRI

Optical microscopy

Electron microscopy

3D whole-brain histopathology


Material and methods

6GdR ISIS, 23 juin 2015 - T. Delzescaux

Experimental designs

7

• 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?


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


Section to sectionco-alignement

Spatial reference

Image stacking

Series 1 Series n

Histological imagesBlock-face

photographs…

Histological images stacks

3D reconstruction

9

Cresyl violet volume6E10 IHC volume IgG IHC

volumeBlock-face photographs

volume (spatial reference)


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


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


6E10 IHC segmented volume

IgG IHC segmentedvolume

Mouse brain altas

Applications


13

Mouse model characterization

→ Amyloid load deposition +++ in cerebral cortex and hippocampal region

→ Amyloid deposition detected in basal ganglia and white matter tracts


14

Immunotherapy evaluation

→ Amyloid load is close to none in PS1 mice

→ 13C3a lowered amyloid load in APP/PS1 mice.


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


Validation and comparison

with

current standards


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


Perspectives and

preliminary results


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


Multiple markers 3D histopathology [1/3]

Information

acquired at

cellular level

Analysis

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From mesoscopic to microscopic scale (1/4)


Multiple markers 3D histopathology [2/3]

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(Zhenzhen YOU)

Mouse brain section (Iba1)

Microglia cell

New challenges to adress: cell indidualization, big data processing, etc...


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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