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Mouse 101
Histology/Histopathology: Considerations and Applications in Biomedical Research
Hibret Adissu, D.V.M., DVSc, Ph.D. Diplomate, ACVP
Investigative Pathologist/Staff Scientist
Molecular Pathology Unit
Laboratory of Cancer Biology and Genetics
May 29, 2018
Comparative Biomedical Scientist Training Programhttp://nih-cbstp.nci.nih.gov
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▪ Comparative Biomedical Scientist Training Program
▪ Animal Model Credentialing and Preclinical Translation - Comparative
Oncology Research
▪ Computer Assisted Diagnostics and Computational Image Analysis
Laboratory of Cancer Biology and Genetics
The Molecular Pathology Unit
John Hickerson,Program Analyst
Jennifer Dwyer, BS, MS
Biologist
Shelley Hoover, BS, HT, HTLBiologist/
Lab Manager
Munish Puri, PhDDigital Pathology
Fellow
Mark SimpsonDVM, PhD, DACVP
Senior Scientist/Director -MPU
Bih-Rong, Wei, PhDStaff Scientist
NCI Center for Cancer Research
Belen Hernandez, DVMMRSP Fellow
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NIH-University Training Partnership
Develop DVM/PhD clinician-scientists
Combined veterinary pathology residency and PhD
➢ Investigative comparative pathology➢ Mouse pathology/Human pathology➢ Ph.D. dissertation research in an
intramural laboratory at the NIH
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Main purposes of this presentation
➢Discuss practical considerations for use of histology/histopathology to support validation of mouse models of human disease
➢Communicate the importance of proper planning to maximize the data generated from pathology
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Histology/histopathology in mouse models –
Universal working goals
➢ Define phenotypes: gain insight into genomic function
and disease mechanisms
➢ Provide tissue for molecular discovery and confirmation
➢ Relate model to natural disease (extrapolate)
➢ Relate experimental endpoints to in-life course (prognosticate)
Consider the tissue as both product and source
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Histology Histopathology
• Study design
• Study execution
• In-life observations
• Specimens procured/preserved
• Specimens analyzed/processing, assay
• Data acquisition,interpretation/extrapolation
The end product or what you can achieve out of histology/histopathology is influenced by many factors
• Appropriate model
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• Mimics host biology
• Represents disease
• Permits questions to be asked and answered
• Predictive
An appropriate animal model…
Can be influenced by…
• Genetics
• Environment
• Health status
The goal of model validation is to determine if the experimental system recapitulates the human disease—genetically, mechanistically, and morphologically at the level of the cell, tissue, organ, organ system, and organism (Brayton and Treuting, 2012)
Animal Model Validation
Cory Brayton C and Treuting PM. 2012 Phenotyping.
Comparative Anatomy and Histology. A Mouse and Human Atlas
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In-life observations and pathology
Metabolism
Growth analysis
Neurobehavioral
testing
Electrocardiography
(ECG)Reproductive
assessment
Ophthalmology
Morphology
Immunology
Clinical laboratory
tests
Pathology
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For any translational research pathology plans should be developed early in experimental design
Unfortunately, too often the thoughts of pathology come later…
At what point to think of histology and pathology studies?
Avoid viewing histology/histopathology as a fixed,static, dehydrated, and stained tissue adhered to glass
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The Mouse ‘Phenotype’
http://newraycom.com
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WT Rsph9-/-
Pathology provides morphological basis for in vivo findings/in-life phenotypes – validates model
Phenotype-Preweaning lethality-Dilated ventricles (MRI)
Histopathology-Dilated ventricles (hydrocephalus)-Suppurative rhinitis
Lesions consistent with a defect in motile cilia (ciliopathy)
Radial spoke head protein 9 (RSPH9)is associated with Primary Ciliary Dyskinesia 12 CLCD12) in humans (OMIM #612650)
Dickinson et al. Nature. 2016, 537:7621):508-514
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Pathology detects subtle abnormalities in the absence of in-life phenotype and predicts progression of disease
Subtle/equivocal or ‘absence’ of in-life phenotype is common in mutant mice. In many of these cases, histopathology is the only assay revealing a phenotype
Age = 4 month
Oval cell and biliary hyperplasia
Genotype: Hepatocyte specific KO of a tumor suppressor gene
In-life phenotype: None detected (at 16 weeks of age)
Age = 12 month; Hepatocellular
carcinoma with pulmonary metastasis
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Digital Pathology
The capture, storage, and interpretation of pathologic specimens using images in digital file formats.
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Histology Histopathology
• Study design
• Study execution
• In-life observations
• Specimens procured/preserved
• Specimens analyzed/processing, assay
• Data acquisition,interpretation/extrapolation
The end product or what you can achieve out of histology/histopathology is influenced by many factors
• Appropriate model
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Sample collection and preservation
Systematic sampling➢ Influenced by anticipated downstream analyses
➢ Plan to triage the collection of clinical laboratory, microbiological, molecular, and histologic samples according to the importance of the sample to the study
➢ select a specimen from an area that illustrates normal tissue along with the adjacent lesion(s)
➢ Rapidly move to preservationFixatives (formalin and others)
Freeze in OCT - for frozen sectioning
Snap frozen
Store in RNAlater
Karwan A.Moutasim et al., Diagnostic Histopathology. 2017. 23:243-249
Normal
Lesion
Normal
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Tissue handlingThe Dark Knight
Returns
‘dark neuron’ artifact
Jortner BS. 2006. The return of the dark neuron.
A histological artifact complicating contemporary
neurotoxicologic evaluation. Neurotoxicology
Avoid bruising tissue with forceps
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Inflation of mouse lungs by way of airway perfusion of fixative (insufflation)
Prevent collapse of alveoli
Caution:
avoid overinflationKnoblaugh S, Randolph-Habecker J, Rath S. Necropsy and Histology. Comparative Anatomy and Histology A Mouse and Human Atlas
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Underinflation of lung
Poorly inflatedOptimally inflated
✓
✗
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Overinflation
Over inflated
Properly inflated
Excessive inflation
Optimal inflation
Edema, emphysema?
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➢ Underfixing or overfixing can compromise histology, immunohistochemistry - costly
➢ Sufficient fixative (1:10 V:V) for immersion fixation -minimum 12-24 hours (room temperature is best)
➢ 10% Neutral buffered formalin (NBF) penetrates tissue at 0.78mm/hr
➢ Large pieces : cut to smaller pieces to allow proper infiltration of fixative for optimal fixation.
➢ “No thicker than a nickel, no larger than a postage stamp”
➢ Sometimes need whole body perfusion ➢ May compromise microbiology, photography
Tissue Fixation
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Formalin solution, 10% NBF (4% formaldehyde in PBS)▪ Most commonly used fixative for light microscopy (prolonged storage)▪ Not suitable for electron microscopy (methanol in formalin may coagulate
cellular components alter fine tissue ultrastructure).▪ Autofluorescence (increases with prolonged exposure)
Paraformaldehyde (polymerized formaldehyde)▪ Must be made fresh before each use (ie converted to formaldehyde
solution)▪ Does not contain stabilizer methanol preferred to formalin for electron
microscopy
Glutataldehyde▪ Choice of fixative for EM.
Tissue Fixation
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Curtesy – Dr. Susan Newbigging, Toronto Center for Phenogenomics, Toronto, Canada
Inadequate fixation
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The Good, The Bad, and The Ugly
Tissue ‘fixation’
http://vnmanpower.com/en/hr-management-the-good-the-bad-and-the-ugly-bl221.htmlSlide credit: Dr. Tyler Peat, NCI/CBSTP
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Factors Influencing immunohistochemistry
Molecular Oncology, Volume 8, Issue 4, 2014, 783–798
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Examples quantitative digital pathology analysis applications in research
Tumor Burden (H&E stain);Necrosis (H&E Stain);Cytodifferentiation;
Nuclear; Positive pixel count; Co-localization
Cytonuclear
Color Deconvolution
Machine learning pattern recognition (can be combined with other algorithms)
Cell Proliferation; ApoptosisImmune cell traffic
Protein of interest expressione.g. signal transduction
Extent of Fibrosis;Cell / matrix development
T-Cell Infiltration; Invading Front Spatial analysis- Infiltration
Image analysis algorithm
Microvessel densityAngiogenesis
End point/output
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13.7% Tumor
Pattern recognition analysis to quantify metastatic burden in the lung
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Cytonuclear analysis to quantify apoptosis or proliferation in metastatic tumor in the lung
Immunohistochemistry quantification
Cell proliferation (Ki67); apoptosis (Cleaved caspase-3)
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Total area of tissue =123.43mm2
Cartilaginous differentiation area = 6.84 mm2 (5.54%)
1.5X
15.2X
H&E Stained Slide Image Pseudo-color Mark up
Osteoid
Cartilage
Undifferentiatedtumor
Osteoid
Cartilage
cFOS-SOX9 Axis Reprograms Bone Marrow-Derived Mesenchymal Stem Cells into
Chondroblastic Osteosarcoma. He Y et al. Stem Cell Reports. 2017, 8:1630-1644
Chondro-osseous
Undifferentiatedtumor
Undifferentiatedtumor
Pattern recognition algorithm to identify/quantify tissue differentiation in a sarcoma
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Wei BR, et al. Pigment Cell Melanoma Res. 2016 PMID: 27463366
Melanomas treated with the
combination treatment had less
nuclear p‐ERK in association with
greater plasma GSK1120212
concentration
V - vehicle
N - BEZ235
G - GSK1120212
C - Combination
GSK1120212 - MEK inhibitor
NVP‐BEZ23 -dual PI3K/mTOR inhibitor
Cytonuclear analysis to assess/quantify target modulation in a cancer model
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Histology Histopathology
• Study design
• Study execution
• In-life observations
• Specimens procured/preserved
• Specimens analyzed/processing, assay
• Data acquisition,interpretation/extrapolation
The end product or what you can achieve out of histology/histopathology is influenced by many factors
• Appropriate model
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Summary➢ Histology/histopathology plays a pivotal role in the
interpretation of in vivo assays and in-life phenotypes
by providing an integrated overview of the disease
processes in the whole organism (‘miniaturizes the
elephant’)
➢The output from histology/histopathology is influenced
by many factors, mainly by proper tissue collection and
preservation
➢Advanced planning is critical to ensure optimal output
from histology/histopathology and subsequent
molecular analysis; hence its contribution to
translational research and its impact on precision
medicine
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The Molecular Pathology Unit
John Hickerson,Program Analyst
Jennifer Dwyer, BS, MS
Biologist
Shelley Hoover, BS, HT, HTLBiologist/
Lab Manager
Munish Puri, PhDDigital Pathology
Fellow
Mark SimpsonDVM, PhD, DACVP
Senior Scientist/Director -MPU
Bih-Rong, Wei, PhDStaff Scientist
NCI Center for Cancer Research
Belen Hernandez, DVMMRSP Fellow
Acknowledgments
Charles Halsey DVM, PhD, DACVP
Pfizer
Susan Newbigging DVM, MSc
The Center for Phenogenomics, Canada
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Resources
• http://nih-cbstp.nci.nih.gov/resources_pathology/index.asp
• A Practical Guide to the Histology of the Mouse, C. Scudamore
• http://www.biocompare.com/Antibodies/
• Comparative Anatomy and Histology. A Mouse and Human Atlas
(https://www.sciencedirect.com/science/book/9780123813619)
• Mouse histology atlas
http://www.deltagen.com/target/histologyatlas/HistologyAtlas.html
• The Anatomy of the Laboratory Mouse. Margaret J. Cook (http://www.informatics.jax.org/cookbook/contents.shtml)
• http://www.ihcworld.com
• https://ncifrederick.cancer.gov/rtp/lasp/phl/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201123/