Comparative Histopathology (Mouse vs Human)mcp.bs.jhmi.edu/sites/default/files/18 BSTP BRayton ComPAth +COD 1 15.pdf · stomach) Ward et al. 2001. Hyalinosis andYm1/Ym2 gene expression

Comparative Histopathology

(Mouse vs Human)Cory Brayton, DVM, Diplomate, ACLAM, ACVP

Director, Phenotyping CoreAssociate Professor, Molecular and Comparative Pathobiology

Johns Hopkins University, School of Medicine733 North Broadway BRB 851

Baltimore, MD [email protected]

• No financial disclosures that I know of…

• All opinions expressed and implied in this presentation are solely those of Dr. Brayton.

• The content of the presentation does NOT represent or reflect the views of Johns Hopkins University or Johns Hopkins Health system…

Disclosures

2

Aims• GET INTERESTED ….

• Appreciate useful similarities.

• Appreciate research relevant differences.• Appreciate pathology & use it ….

• DO GREAT SCIENCE, REPORT IT USEFULLY!

MISSION : Comparative Histopathology in 45MIN!?

3

BetterResearch Better Health

4

Discussion Plan: MICE v human1. Getting practical ….2. LIZ M3. THYMUS Finding it

• ATHYMIC? Really? 4. Bone marrow – decal or no

• Quantifying cellularity • Spin vs blowout for cytology

5. Lungs INFuse vs PERfuse• Mouse lung = distal human airways• PINK Stuff AMP/PcP – vs PAP

6. Kidney useful sections• Glomeruli Cuboidal metaplasia • Hyaline glomerulopathy vs GN etc

7. Salivary and lacrimal glands • Sexual dimorphisms• Irradiation

8. Pancreas 1 slide • big islets in fat mice,

9. GI tract Sections vs Rolls • Forestomach relevant to esophagus • Tumors & IBD models• Appreciate the Flora

10.REPRO 1 slide• Prostate comparison

11.HEAD Decal vs Brain removal • Its not all in the brain• eyes, ears, • nose, dental disease • molar vs incisor irradiation etc genotoxins

Resources1. Brayton 2013 rev: Spontaneous diseases in commonly used mouse strains and stocks

http://phenome.jax.org/projects/Brayton12. Brayton et al. 2012. Pathobiology of aging mice and GEM: background strains and experimental

design. Vet Pathol. 49(1):85‐105.3. Brayton et al. 2014. Analysis of Phenotype. In Transgenic Animal Technology… Pinkert, Elsevier: 431‐488. (necropsy protocol reference) Lab manual at http://mcp.bs.jhmi.edu/me680712‐phenotyping‐functional‐genetics

4. DORA Diseases of Research Animals http://dora.missouri.edu/5. Frith and Ward. 1988. A Color Atlas of Neoplastic and Non Neoplastic Lesions in Aging Mice.

http://www.informatics.jax.org/frithbook/ also print on Demand from http://store.cldavis.org/6. McInnes. 2011. Background Lesions in Laboratory Animals, Elsevier 7. MGI Mouse Genome Informatics SEARCH GENES etc info http://www.informatics.jax.org/

IMSR ‐ FIND MICE AND ES CELL LINES http://www.findmice.org/index.jsp Mouse (rat) and Gene NOMENCLATURE & apply for a laboratory code

http://www.informatics.jax.org/mgihome/nomen/index.shtml MPD‐Mouse Phenome Database mouse data and protocols http://phenome.jax.org/ MTB Database & tumor frequency grid http://tumor.informatics.jax.org/mtbwi/index.do

8. NTP NON neoplastic lesion atlas http://ntp.niehs.nih.gov/nnl/9. Pathbase: European mutant mouse pathology database http://www.pathbase.net/10. RENI Tissue trimming guide + INHAND terminology and diagnostic criteria

http://www.goreni.org/11. Scudamore, Ed. 2014. A Practical Guide to the Histology of the Mouse, Wiley Blackwell.12. Treuting, Dintzis, Montine. 2017 Comparative Anatomy and Histology 2nd Edition Mouse, Rat, and Human Atlas https://www.elsevier.com/books/comparative‐anatomy‐and‐

histology/treuting/978‐0‐12‐802900‐8

• PERFUSED MICE + LABELS/ID Can fit in tubes or appropriate sealed containers for years(?)

• Fixed tissues may outlast the container ….

• Safety concerns? With fixatives?

• Don’t discard or Freeze mice… including controls

… ?!

6

2018 BSTP Comparative Histopath / Common COD

[email protected] 2018 Page 1 of 24

7

A 10 slide mouse protocol

• Perfusion & Dissections by Nadine Forbes McBean;

• Slides by Bonnie Gambichler

• Slide scanning courtesy of Flagship Biosciences

• Ref: Brayton et al. 2014. Analysis of Phenotype. Transgenic Animal Technology, A Laboratory Handbook. Pinkert, Elsevier: 431‐488. https://www.elsevier.com/books/transgenic‐animal‐technology/pinkert/978‐0‐12‐410490‐7

• Download manual / protocol http://mcp.bs.jhmi.edu/me680712‐phenotyping‐functional‐genetics

1. Heart, Sternum, Thymus,Tongue

6. GI –Cross sections

2. Lung, Trachea, Thyroid, Esophagus

7. Liver, Gall bladder, Spleen

3. Kidneys (R cross, L Long), Adrenal

8. Repro (Female)

4. Salivary glands, Lymph nodes (Female)

8. Repro (Male)

4. Salivary glands, Lymph nodes (Male)

9. Skin

5. Pancreas, Mesentery

10. Head (post decal)

15mm

15mm

15mm

15mm

15mm

15mm

15mm

15mm

15mm

15mm

15mm

15mm

1 Heart Sternum ThymusTongue

8

1 Heart Sternum ThymusTongue (perfused)

9

• What to look for by slide/tissue (basics)

Treasure Hunt!!

http://mcp.bs.jhmi.edu/me680712‐phenotyping‐functional‐genetics

Thymus in situ

11

• Record thymus size or wt.• OR NT = no tissue

• Sternum removed• Salivary glands removed

12

THYMUS• ECTOPICNear salivary GL Near thyroids

Athymic ?NSG …. ‘Lymphocyte hypoplasia’ (aplasia)



13

MICE vs human Marrow

• NON Decal Sternum Better for IHC? Technical – Beware of Surface decal

• DECAL Head, Leg Spine etc Useful for assessing other lesions Some IHC works

• Cellularity Automated on H&E: Koslowski et al 2018

Human

• OUCH

• Ischium, sternum Core aspirates Cytology

14

Marrow

Sternum• NOT decalcified

• Cellularity Hi , Mod, Lo

• Trilineage ?

• M:E ? Myeloid: Erythroid

• Pigment

• Crystals

15

Marrow

Sternum• NOT decalcified

• Cellularity: Hi• Trilineage ?

• M:E ~4

• Pigment

• Crystals

16

Marrow • Leg, knee • Decal Formic acid Skin removed

• Lymphoma

17

MICE vs human Marrow

• Cytology Cytospin FACS

• Femur Blow out

• Femur Centrifuge

Human

• OUCH

• Ischium, sternum Core aspirates Cytology

2 LUNG Etc

• Lungs• Trachea• Larynx (+ thyroids)• Esophagus

Mediastinal lymph nodes Thymus remnants Aorta

18Back down (dorsal down) in cassette



2 LUNG – perfused + infused

19 20

Lethal Respiratory diseaseIn immunodeficient mice

• Sudden Death(Found Dead)

• Diffuse consolidation• Interstitial pneumonia

20

MICE vs human Lung

21

CF

NEJM.org

• Mouse lung = distal human airways NO intrapulmonary bronchi (no Cartilage + no submucosal glands) less pH change less lung dz in CF models

• Mice lungs fit on 1 slide Left lung is simpler (like Human)

• Strain variations Airway anatomy (Mitzner et al) Acidophilic Macrophage Pneumonia (Swiss, B6, 129 etc)

o YM1 accumulationso Increased with various infections allergens

TUMORS A!, Balb, 129 FVB/N etc

MICE vs human Lung

[email protected] 22

H&EEM

PAS

IHC

https://www.nejm.org/doi/full/10.1056/NEJMra023226 Trapnell et al 2003

• Pulmonary Alveolar Proteinosis human

• GEM model HAD Lung disease ! &

• GREAT LESIONS !!!

• Do we have the right lesion?Just describe the phenotype and take a picture for the manuscript …

LUNG PINK stuff human

23

LUNG PINK stuff MOUSE



25

LUNG PINK stuff MOUSEAcidophilic Macrophage ‘Pneumonia’aka Acidophilic Crystalline ‘Pneumonia’

• NOT PAP• Cause of Death in susceptible strains: 129, B6/129, Swiss

• Extensive consolidation

• Research confounder: Chitinase like proteins upregulated in asthma, infections

• OR incidental finding Scattered Acidophilic Macrophages or Crystals

25 26

PAP

• Pulmonary Alveolar proteinosis

• EM myelin figures (lamellar bodies)

• Not crystals

• Induced model of alveolar proteinosis in rat model

• Vijëyaratnam & Corrin, 1972

Chil3 (Ym1), YM2 chitinase like proteinsin Hyalinosis & Acidophilic Macrophage Pneumonia

Haines et al. 2001. Toxicol Pathol. 29(6):653‐61. Pathology of aging B6;129 mice.

Hyalinosis + extracellular crystals multiple sites (respiratory, gall bladder, stomach)

Ward et al. 2001. Hyalinosis and Ym1/Ym2 gene expression in the stomach and respiratory tract of 129S4/SvJae and wild‐type and CYP1A2‐null B6, 129 mice. AJP Hyalinosis (eosinophilic cytoplasmic change) in glandular stomach, respiratory tract,

bile duct, and gall bladder of B6,129 CYP1A2‐null & wild‐type mice Also in both sexes of background 129S4/SvJae strain (WT) Ym1 in normal and abnormal NOSE olfactory + respiratory, lung, marrow, stomach Ym2 in hyaline Stomach lesions

Nio et al. 2004. Histochem Cell Biol.121(6):473‐82. Cellular expression of murine Ym1 and Ym2, chitinase family proteins, as revealed by in situ hybridization and immunohistochemistry.

chitinase family proteins, widely distributed, bind GAG’s such as heparin/heparan sulfate.

Ym1 is a macrophage protein produced in parasitic infections…o Lung ‐‐Ym1‐expressing cells are alveolar macrophages, Ym1 localized in RER. o Spleen ‐‐Ym1‐expressing cells in red pulp identified as immature neutrophils. o Marrow ‐‐ immature neutrophils lose immunoreactivity with maturation. o Marrow ‐‐ needle‐shaped crystals in macrophages show intense Ym1

immunoreactivity 27

Lung, GMS

LUNG PINK stuff MOUSE

• Next lecture…

29

MICE V human Kidney

Gross R kidney higher/cranial ~14000 Nephrons

Histology

• Glomeruli cuboidal parietal epithelium is more common in males (normal)

• PAS stain for Prox tubules brush border

Function

• Rodents concentrate better >1040 SG Dt More long segment nephrons

• BUN /creat are insensitive biomarkers

• Human R kidney lower ~1 million nephrons/kid Glomeruli cuboidal parietal epithelium is a lesion

[email protected]

3 Kidneys, Adrenals

30



3 Kidneys, Adrenals

31

JUST Cortex…All Segments

Adrenals

32

NNLA KIDNEY

• Cortex, • medulla,

• renal papilla, • renal pelvis

• Medulla outer stripe of outer medulla (OSOM), inner stripe of outer medulla (ISOM), inner medulla

https://ntp.niehs.nih.gov/nnl/urinary/kidney/index.htm

Which is more likely to be male?A B

Cuboidal metaplasia in HUMANS associated alcoholism etc….(Tubule vacuolation more likely in (fat) males) 34

Kidney

34

• Glomerular cuboidal parietal epithelium

• More common in male

69216

Old Mouse Kidneys: PINK STUFF

PINK STUFF in glomeruli Special stains

GN Membranoproliferative

+/‐ glomerulosclerosis

PAS+ thick BM

Small, trichrome ++

Amyloidosis Glomeruli +/‐ interstitial CONGO+ (Tri)

Hyaline glomerulopathy PAS+ (Tri)

Fibrocollagen glomerulopathyGM? TRICHROME++

35 36

PINK STUFF

• 22mo, F

• B6.129P2‐Il10(tm1Cgn)/J

Special Stains

• PAS• Trichrome

• Congo Red




37

22mo F Mouse

• PAS

[email protected]

22mo F Mouse

• Trichrome

• Not exactly sclerosis

• Tubules look pretty good

[email protected]

39Hoane et al . Copyright © 2016 Society of Toxicologic Pathology

MICE Hyaline glomerulopathy

• Enlarged glomeruli

• PAS positive

• Massons trichrome light blue‐purple

• Congo Red negative

• Mouse ‘NOS’

• c 1983

Special Stains

• PAS• Trichrome

• Congo Red

PINK STUFF


41

• Diagnosis/es

• Special stains

[email protected]

Primary systemic AL

AL monoclonal IG light chains From clonal plasma cells

kidneys, heart, liver

Hu NHP dog

Secondary reactive SAA

serum amyloid A Chronic inflammation

Gut liver kidney etc

Many mammals birds

Transthyretin TTR

autosomal dominant (+senile)

Liver, heart,kidney(Rx transplant)

Hu

Apolipoprotein A1Apoa1

autosomal dominant (‘senile’)

Heart, liver kidney

Hu NHP mice etc?

Diabetic amyloidIAPP

Islet amyloid precursor protein

Islets Hu NHP cat dog Etc Obese Type 2dm

Amyloidoses




43

Mouse Amyloidosis

• Gut – Intestinal amyloid • Distal SI

• Kidney – Renal amyloid Glomeruli > interstitial

• Liver – Hepatic amyloid Etc.

o Adrenalo Salivary glandso Tongue o Hearto Stomacho Testes

Weight loss & Wasting‘Premature Aging’ phenotype

e.g. in SAMP Senescence Accelerated Mice

43

Malabsorption

protein loss

size, function

Mouse Amyloid

• WERE COMMON complicators of chronic studies less ‘clean’ conditions

1. Reactive, Secondary, AA – SAAoSAA serum amyloid A component

□ Acute phase response protein

o chronic immune stimulation –GI, kidney, spleen

2. Senile, ASSAM amyloid – Apoa2c >> Apoa2aoApolipoprotein A2 (part of HDL )oAlleles a,b,c,d,e

44

45

Apoa2 gene/locusallele Strains

ApoA2a AKR, BTBR, C57BL/6J, C57BL/10, DBA/2SAM‐P3, SAM‐P8, SAM‐R2

ApoA2b 129S1, BALB/CJ, BALB/cByJ, C3H/, CBA, FVB/NJ, MRL, NZW etcSAM‐P6, SAM‐R1

ApoA2cA/J, SJL/J etcSAM‐P1, SAM‐P2, SAM‐P7, SAM‐P9,

ApoA2d PERA, WSB

ApoA2e SPRET

• c allele EARLY ASSAM onset in liver spleeno ‘aging phenotype’

• a allele ‐ later onset in gut, lung, tongue etc.• b allele –QTL associated with higher HDL level

o Summarized from Higuchi & al 1991; Wang & al 2004

45

Pink Stuff in Glomeruli

46 8 mo, female, B6C3Fe-a/a-Cola2oim/oim

Beware of GEM…

478mo, female, B6C3Fe-a/a-Cola2oim/oim

Fibrocollagen glomerulopathy 48

MICE vs human Salivary / Lacrimal

• Fit on 1 slide Ear to ear – includes Submandibular Sublingual Parotid [Exorbital lacrimal glands] Lymph nodes

• Expect Sexual dimorphism

[email protected]



4949

Salivary Glands, associated Nodes

A

B

Which is Male ?

A or B ?

1. Submandibular2. Sublingual

3. Parotid (exorbital lacrimal)

4. Lymph nodes

MICE vs human Pancreas• Fit on 1 slide With Lymph nodes etc Fat etc

• Expect Zymogen depletion in sick mice Bigger islets in fat mice


5 Pancreas etc

Pancreas, mesentery, lymph nodes Fix in in cassette at dissection OR lay flat on paper for fix

51

5 Pancreas etc

52

• Obesity

• Insulin resistance• Type 2 DM• Big islets

PANCREAS

53

• PANin, PDAC

• KPC • Kras + p53

PANCREAS

54



MICE vs human GIT (gut)

• ~ 5 Pieces /slide • Closed Rolls (~3 slides) • Open Rolls

• I like content Friends/frenemies Parasites Dysbiosis

• Forestomach ~esophagus

• Cecum ~appendix


6 GIT

56

Most labs won’t accept so many pieces..Some labs remove gut content….

6 GIT• Too many pieces….

57

Swiss roll options…

• ‘Closed’ I like gut content

• ‘Open’ allows gross assessment of lesionsThen assess selected lesions

58

59

GIT

59

• Closed roll Content Exudate Pseudomembrane Intact mucosa

• Open roll prettier…

MICE vs human GIT (gut)• Microbiome = microbial genomes

• Microbiota = all the agents\

• Not just bacteria Viruses fungi protists metazoan parasites

• NOT just feces Important variations by site




7 Liver, Spleen

61

Med

Med

7 Liver, Spleen

62

8 REPRODUCTIVE

63

May NOT fit in 1 cassette …• Age• Parity • Diet ‐ phytoestrogens ?

64

PROSTATE

• Human prostate 1 “lobe” 3 zones: central, transitional, peripheral Most PCa in peripheral zone ~75% of prostate

• Mouse prostate 4lobes Anterior = coagulating gland Dorsolateral – closest to human Peripheral zone?

64Valkenburg & williams 2011https://www.ncbi.nlm.nih.gov/pubmed/22111002

Shappell et al. Cancer Res 2004;64:2270-2305©2004 by American Association for Cancer Research

Mouse prostate

VP, ventral prostate; LP, lateral prostate; DP, dorsal prostate; SV, seminal vesicles;CG, coagulating gland or anterior prostate;

DD, ductus deferens; UB, urinary bladder. Horizontal black line indicates the level of transverse sectioning thru urethra

1) urethra in cross‐section;2) paired ductus deferens 3) Paired ampullary glands 4) Ventral prostate;5) lateral prostate;6) Dorsal prostate has fibromusc stroma

9 Skin • Nice ribbons – note Fat, mammary, preputial or clitoral glands

66



MICE vs human Head

• Fit on 1 slide ~5 Decal sections Eyes Ears Nose Harderian gland

• Expect


10 Decalcified Head

1. Cerebellum medulla□ Front down

2. Ears hippocampus, midbrain, pituitary

□ Front down

3. Forebrain, molars□ Back down

4. Eyes, olfactory bulbs□ Back down

5. Nose □ Back down

68

10 Decalcified Head

69

• Fit on 1 slide ~5 Decal sections Eyes Ears Nose Harderian gland

• Expect

Treasure hunt! Head

70

Lots of Info NOT in the brainhttp://mcp.bs.jhmi.edu/me680712‐phenotyping‐functional‐genetics

71

129 (& BALB/c) Brain phenotype • NO corpus callosum AKA Acallosity

• Not every mouse

71varies with strain and supplier of the mice Wahlsten 1982

Rolling, Spinning, Head tilt(vestibular)

Dizzy mice –GEM Model of vestibular syndrome?

• Consider:1. Otitis?2. Arteritis ?3. Tumors?4. Other?

72



'Neurologic' #2: Rolling, Spinning, Head tilt (vestibular)

Dizzy mice –GEM Model of vestibular syndrome?

• Consider:1. Otitis?2. Arteritis ?3. Tumors?4. Other?

73

Mice

• Stroke…

Mouse Pituitary Tumors …

• Adenomas of pars distalis are more common than of pars intermedia.

• Aged females.

• Like rat may secrete prolactin

• well‐delineated

• Compressive

• angiectatic or cyst‐like spaces are common.

75 76

Mice Head tumors

77

41086

• Enucleated • Fekete’s fixATIVEAcid alcohol


67189 nu/nu 67188 FVB/N



79

70274 NSG 41086 WNL

Perinatal irradiation

80

60635 Rag1 KO

• Otitis

• Rag1 KO Myd88 def

Mice

82

Midline sections can be informative

• Fresh /fixed hemisection

• Midline section between incisor Cut/crunch sphenoid bone ONLY

• Or Fix/decal for hemisection

83

60371 NOG dying

• Spine • Formic acid fix +decal

• No lymphocytes ???

• Lymphoma/Leukemia

EtOH DAMAGES CNS! NO weekend /holiday processing!



129? Teratomas (Ter), lung tumors, acallosity, AMP, …A/J Lung tumors, anomalies, amyloid, muscular dystrophyAKR Thymic Lymphoma…BALB/c Plasmacytoma etc tumors, heart dz, acallosity, kill each otherC3H TUMORS ‐ Mammary, Liver

C57BL/6 Microphthalmia, Hydrocephalus , MUD, Osteoporosis, Presbyacusis, Amyloidosis, AMP, Tumor resistant? …

DBA Deaf, seizures, glaucoma, autoimmuneFVB/N? Blind, seizures, mammary/pituitary dzNOD Diabetes, immunoweirdSJL/J Lymphoma, muscular dystrophy, kill each otherDEAF C57BL/6, BALB, DBA, etcBLIND rd1 C3H, CBA, SJL, SWR, FVB

STRAIN MATTERS.Some Phenotypes in ‘Normal’? (+/+) Mice

85

WILDA, BALB, C3

DBA

129

C57, C58

Which strain to represent all mice?& humans?

Group 1: Bagg albino derivatives

Group 2: Swiss

Group 3: Japanese & New Zealand inbred

Group 4: Abbie Lathrop’s C57/58

Group 5: Castle's 129 etc

Group 6: CC Little's DBA & related

Group 7: WILD DERIVED

Petkov et al 2004. An efficient SNP system for mouse genome scanning elucidating strain relationships. Genome Res. 2004 Sep;14(9):1806‐11.

Nomenclature – mission critical in scientific communication…• Correct strain names identify strain(s) of origin, and where it has been, and came from

• Correct mutant names tell a lot about the mice & mutations

• Incorrect names compromise communication, reproducibility, translational research

87

USE CORRECT NAMES. REQUIRE CORRECT NAMES when reviewing & editing. Get a Lab Code if you make or breed mice Its FREE $ & EASY! USE THIS SITE:

http://www.informatics.jax.org/mgihome/nomen/index.shtml

RESOURCE !!

Prevalence of sexual dimorphism in mammalian phenotypic traits. Nat Commun. 2017 Jun 26;8:15475. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490203/

How many authors to convince a scientist?

• Karp et al (159 collaborators in IMPC) : Mason J, BeaudetAL, BenjaminiY, Bower L, Braun RE, Brown SDM, Chesler EJ, Dickinson ME, FlennikenAM, Fuchs H, Angelis MH, Gao X, Guo S, Greenaway S, Heller R, HeraultY, Justice MJ, Kurbatova N, Lelliott CJ, Lloyd KCK, Mallon AM, Mank JE, Masuya H, McKerlie C, Meehan TF, Mott RF, Murray SA, Parkinson H, Ramirez‐Solis R, Santos L, Seavitt JR, Smedley D, SorgT, Speak AO, Steel KP, SvensonKL; International Mouse Phenotyping Consortium, Wakana S, West D, Wells S, Westerberg H, Yaacoby S, White JK. bataY, Suzuki T, Tamura M, Kaneda H, FuruseT, Kobayashi K, Miura I, Yamada I, Tanaka N, Yoshiki A, Ayabe S, Clary DA, Tolentino HA, Schuchbauer MA, Tolentino T, Aprile JA, Pedroia SM, Kelsey L, Vukobradovic I, Berberovic Z, Owen C, Qu D, Guo R, Newbigging S, Morikawa L, Law N, Shang X, Feugas P, Wang Y, EskandarianM, Zhu Y, Nutter LMJ, Penton P, LaurinV, Clarke S, Lan Q, Sohel K, Miller D, Clark G, Hunter J, Cabezas J, BubshaitM, Carroll T, Tondat S, MacMaster S, Pereira M, Gertsenstein M, DanismentO, Jacob E, Creighton A, Sleep G, Clark J, Teboul L, Fray M, Caulder A, Loeffler J, Codner G, Cleak J, Johnson S, Szoke‐Kovacs Z, RadageA, MaritatiM, Mianne J, Gardiner W, Allen S, Cater H, Stewart M, Keskivali‐Bond P, Sinclair C, Brown E, Doe B, Wardle‐Jones H, Grau E, Griggs N, Woods M, Kundi H, Griffiths MND, KippC, DT, VancollieVE, Pearson SA, Gates AS, Sanderson M, Shannon C, Anthony LFE, Sumowski MT, McLaren RSB, Swiatkowska A, Isherwood CM, Cambridge EL, Wilson HM, Caetano SS, Mazzeo CI, Dabrowska MH, Lillistone C, Estabel J, Maguire AKB, Roberson LA, Pavlovic G, Birling MC, Marie WD, Jacquot S, Ayadi A, Ali‐Hadji D, Charles P, André P, Le MarchandE, El Amri A, Vasseur L, Aguilar‐Pimentel A, Becker L, Treise I, Moreth K, StoegerT, AmarieOV, Neff F, WurstW, Bekeredjian R, Ollert M, Klopstock T, Calzada‐Wack J, Marschall S, Brommage R, Steinkamp R, Lengger C, Östereicher MA, Maier H, Stoeger C, Leuchtenberger S, YildrimA, Garrett L, Hölter SM, Zimprich A, SeisenbergerC, Bürger A, Graw J, EickelbergO, Zimmer A, Wolf E, Busch DH, Klingenspor M, Schmidt‐Weber C, Gailus‐DurnerV, Beckers J, Rathkolb B, Rozman J. Melvin DG, Raj NPS, Holroyd SA, Gannon DJ, Alcantara R, Galli A, Hooks YE, Tudor CL Green AL Kussy FL Tuck EJ Siragher EJ Maguire SA Lafont

SEX MATTERS TOO.

88

89

SEX MATTERS too.

AUTOIMMUNE• Graves• Hashimoto• RA• SLE• T1DM

INFECTIOUS

• HIV• Flu• Toxo• Legionella• Malaria• Zika

89

NON repro CA • Bladder• Bowel• Kidney • Leukemia• Liver• Lung• Melanoma• Esophagus• Stomach

• Ebola• MERS• HepB• Campy• Schistosomiasis• Amebiasis• Aspergillosis

Adapted f rom Chervonsky 2018Klein & Flanagan 2016

COMMON CAUSES OF UNEXPECTED DEATH

(in research mice)Cory Brayton, DVM, Diplomate, ACLAM, ACVP

Director, Phenotyping CoreAssociate Professor, Molecular and Comparative Pathobiology

Johns Hopkins University, School of Medicine733 North Broadway BRB 851

Baltimore, MD [email protected]



•Cause of Death •Contributing Causes of Death

•Causes of attrition Euthanasia for welfare reason

COD/CCOD

92

Discussion Plan COD/CCOD EMPHASIS UNintended / unexpected consequences of

1. Experimental manipulations – Tx: Bleeding + injections Rx: Estrogen Rx: Antimicrobials

o DOX etc

Rx: Irradiation Rx: Humanizing

2. Common microbes especially in immune deficient mice Pneumocystosis Skin lesions

o MUD, Abscesseso C bovis,

MKPV EBV

Also • PUPs• MUD

Aims• CONSIDER THE CONSEQUENCES of

Common research manipulationsCommon microbial agents

• Assess unexpected (& expected) morbidity/mortalityi.e. use pathology….

• DO GREAT SCIENCE, REPORT IT USEFULLY!

MISSION : Common COD in 30MIN!?

93

BetterResearch Better Health

Resources1. Brayton 2013 rev: Spontaneous diseases in commonly used mouse strains and stocks

http://phenome.jax.org/projects/Brayton12. Brayton et al. 2012. Pathobiology of aging mice and GEM: background strains and experimental

design. Vet Pathol. 49(1):85‐105.3. Brayton et al. 2014. Analysis of Phenotype. In Transgenic Animal Technology… Pinkert, Elsevier: 431‐488. (necropsy protocol reference) Lab manual at http://mcp.bs.jhmi.edu/me680712‐phenotyping‐functional‐genetics

4. DORA Diseases of Research Animals http://dora.missouri.edu/5. Frith and Ward. 1988. A Color Atlas of Neoplastic and Non Neoplastic Lesions in Aging Mice.

http://www.informatics.jax.org/frithbook/ also print on Demand from http://store.cldavis.org/6. McInnes. 2011. Background Lesions in Laboratory Animals, Elsevier 7. MGI Mouse Genome Informatics SEARCH GENES etc info http://www.informatics.jax.org/

IMSR ‐ FIND MICE AND ES CELL LINES http://www.findmice.org/index.jsp Mouse (rat) and Gene NOMENCLATURE & apply for a laboratory code

http://www.informatics.jax.org/mgihome/nomen/index.shtml MPD‐Mouse Phenome Database mouse data and protocols http://phenome.jax.org/ MTB Database & tumor frequency grid http://tumor.informatics.jax.org/mtbwi/index.do

8. NTP NON neoplastic lesion atlas http://ntp.niehs.nih.gov/nnl/9. Pathbase: European mutant mouse pathology database http://www.pathbase.net/10. RENI Tissue trimming guide + INHAND terminology and diagnostic criteria

http://www.goreni.org/11. Scudamore, Ed. 2014. A Practical Guide to the Histology of the Mouse, Wiley Blackwell.12. Treuting, Dintzis, Montine. 2017 Comparative Anatomy and Histology 2nd Edition Mouse, Rat, and Human Atlas https://www.elsevier.com/books/comparative‐anatomy‐and‐

histology/treuting/978‐0‐12‐802900‐8

COD in PUPSto identify by / at weaning:

1. Hydrocephalus

o Later onset not evident at weaning □ E.g. sporadic in MRL etc.

2. Malocclusion / Incisor overgrowth

o Cull or trim o Breed??

3. Microphthalmiao Expect this in C57BL – but it won’t kill themo Especially? Right eye in females

95

HydrocephalusEspecially C57BL

96



97

Malocclusion , incisor overgrowth Secondary Phenotypes

• 6g – 6 weeks old• ‘Developmental delay’ important phenotype? or just not eating (inanition)?

• Early Death97 98Yardeni et al. 2011 – retroorbital injection techniquehttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158461/

COD/CCOD Rx/Technique • Bleeding Retrorbital sinus Facial Vessels

• Injections Retrorbital sinus Vasculature? Intracranial? Intraocular?

• Intracranial? • Intraocular?

COD Retroorbital? injectionAvoid Cecum Usually on Left Can be midline or right Larger with Dysbiosis (antibiotic Rx?) , germ free

• Also on Left Stomach, spleen

• IP Inject Usually on Right

• FYI Uysal et al. 2017. Cecum location (BALB/c) By Ct , Gross

IP Injections

100

101

COD/CCOD Rx

•Estrogen Toxicity /Estrogenization

For ER+ (estrogen receptor positive) tumor cell lines Mortality ‘Watery blood(pancytopenia) Splenomegaly (compensatory hematopoiesis) UTI , urinary tract infections

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Estrogen

• NON estrogenized

• Bearing Breast Ca Cell line (ER neg)

• (‘normal’ marrow)

• DYING MICE

• ESTROGENIZED

• Bearing Breast Ca Cell line ER positive

• Hematopoietic Toxicity

• Bone proliferation

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103

COD/CCOD Rx

• Doxycycline For conditional expression Long term effects?

Stomach mass:

Antimicrobial Expect dysbiosis

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COD/CCOD Rx

• Doxycycline For conditional expression Long term effects?

Alopecia, thickened skin:

Antimicrobial Expect dysbiosis

Clavamox Ameloblast damage hypomineralized enamel

• Enrofloxacin Cartilage effects in growing animals

• Trimethoprim Sulfamethoxizole Thyroid effects

• Fenbendazole Immunomodulatory Anticancer?

• Streptozotocin/streptozocin (STZ) Diabetogenic, Hepatotoxic Nephrotoxic in humans, mice, rats, NHP

Rx Antimicrobials – other effects

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Mihalaş et al. Copyright © 2015 by Society of Toxicologic Pathology http://journals.sagepub.com/doi/abs/10.1177/0192623315610822

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Mihalaş et al. Copyright © 2015 by Society of Toxicologic Pathology http://journals.sagepub.com/doi/abs/10.1177/0192623315610822

C57BL/6 NOS

• Which is normal?

• Diagnosis/es? • Causes?• Human relevance?

• A• Enamel hypomineralization• amoxicillin/clavulanic acid Rx• Amoxicillin Rx in early childhood may be associated with enamel hypomineralization

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COD/CCOD Rx: Irradiation

TYPES Gamma XR Intravenous

o Alpha/beta

• OFF target effects Developing/proliferating tissues Gut Teeth Bones Etc

Cancer therapyTarget = tumors

‘Conditioning ‘ for GraftsTarget = Innate immunity

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NODscid Mortality post irradiation

• Not just an incidental finding

• Larsen & al. 2006

• Irradiation Literature since 1950’s

• .



109

Rx IRRADIATION Beware: Collateral damage ….• INCISOR • Alpha etc

Ameloblasts?

Odontoblasts?

Odontoblasts

Ameloblasts 110

COD/CCOD Rx

110

• IV alpha particles Kidney What’s missing

67316

111

Rx IRRADIATION Beware: Collateral damage ….

69311

ParotidParotid X lac

X lac

69305, 60301Oversimplified from Mark A. Schroeder, and John F. DiPersio Dis. Model. Mech. 2011;4:318-333 etc © 2011. Published by The Company of Biologists Ltd

COD/CCOD Rxin Grafts/’HUMANIZED ’mice: Graft/ donor Host Cond Rx DZ Primary cell

typePathology?

MAJOR mismatch Often B6 (H2b)Spleen/BM

Various (H2d/b/k/a)

TBI 600‐1300 cGy(+/‐Cy)

Systemic / Lethal10‐50d

UsuallyCD4+/Cd8+

GI bleed death NOS

0 Slower? CD4+/Cd8+ NOS

Minor mismatch e.g. B10.D2

Various e.g. BALB/c (H2b)

TBI 00‐1000cgy

Systemic / LethalNOS or by 80d +/‐ skin

Often CD4+>Cd8+

NOS

XENOHuPBMCetc

NSGRag‐ Il2rg‐NSBm‐

TBI 200‐300cgy

Systemic / Lethal NOS 30‐50d

CD4+ or CD8+ NOS

Acute GvHD in mice – oversimplified….(where’s the pathology?)


ACVP Nat Meeting 2018

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COD/CCOD MICROBIAL

•PneumocystosisP murina

• PINKSTUFF in lungs

• Important cause of death in IMMUNODEFICIENT

• immunoweird

GMS



c 2003

• Fig 1 for PAP manuscript

• NOT PAP

115 116

Respiratory disease

• Don’t forget upper respiratory tract … Rhinitis slide 10 Otitis slide 10

Laryngitis slide 2 Tracheitis slide 2

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How can this affect your research?

MUD = Mouse Ulcerative Dermatitis

A. Animal welfare concernB. Euthanasia for humane reasonsC. Altered behavioral responsesD. Altered immune responsesE. All of the above

• Lymphadenomegaly

• Also had • Splenomegaly

• Leukocytosis

• Etc immune relevant phenotypes Chemokines cytokines

COD/CCOD MUD

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119

MUD Rx: Mouse pedicure

• Pedicure restraint device

• Adams, et al. 2016. A "Pedi" Cures All: Toenail Trimming and the Treatment of Ulcerative Dermatitis in Mice." PLoSOne 11(1): e0144871.

120

COD/CCOD Tumor?

a) Squamous cell carcinoma

b) Model for human head & neck tumors

c) Abscesses



121

COD/CCOD Microbial

Mouse. Head.Diagnosis/es?a) Abscesses

(Botryomycosis)b) Reactive hyperplasia

(lymph node) c) Immunomodulationd) Welfare concerns…

Common Isolates?

122

COD/CCOD Microbial

[email protected]

• ABSCESSES• Common isolates Staphylococci

o S aureus in immune sufficiento S xylosus etc coag neg in immune deficient

P pneumotropica Various commensals in immune deficient

• Myd88 def

• No innate immunity

123Adhikary et al. Copyright © 2017 Laboratory Animals Limitedhttp://journals.sagepub.com/doi/figure/10.1177/0023677217698503?

P pneumotropica

• Bom:NMRI‐Foxn1nu

• Inbred or outbred?

• Diagnosis/es?• Orbital/periorbital abscesses

• C57BL/6JBom

• Inbred or outbred?

• Diagnosis/es?• Subcutis abscess

124Adhikary et al. Copyright © 2017 Laboratory Animals Limited

• Fig 4 Pasteurella pneumotropica

• Outbreak strains = SAME STRAIN

• Clonal outbreaks imply primary role in infection.

•Table 1 Affected mouse strains

• Immune sufficient +

• Immunedeficient

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COD/CCOD Microbial Scruffy & dying mice …

• Corynebacterium bovis Novel HAC? S xylosus? Usually an immunodeficient problem May not be so obvious in haired mice (Facial pattern

Stress? Dehydration Poor Pupsurvival

• PDX survival

Nude mouse RAR

NSG RAR

• Flakey skin + pup mortality Nude, Rag1, NODscid, NSG etc

• Corynebacterium bovis + Novel HAC (+ S xylosus )

• Treatable ? Survivable?

COD/CCOD Microbial Scruffy & dying mice …

Gram positive



127

COD/CCOD MICROBIALKIDNEY DZ

• 2006: 58499• GEM RAG1‐/‐BALB/c & ?

• Related cagematesdied..

61232• Hprt tg nude

• 2009

[email protected]

129

Santagostino et al 2017 : NSG

• 2 INIB in tubule epithelium Large amphophilic Small eosinophilic

• Karyomegaly, tubule degeneration, necrosis

• TEM e‐lucent schmutz

• Serology, PCR, IHC negative for viruses 1 report of positive IHC for a bovine papillomavirus antigen

• Cohoused immune deficient develop similar lesions CD‐1 do not

[email protected]

Roediger et al 2018 CELL


131

What might have been more interesting?

A. BK virus B. JC virusC. MAd1 FL

D. Mad 2 K87E. SV40

F. New /old rat polyomavirus

A useful stain for these agents?

A. ~80% of renal transplant PTB. 10‐20% of renal transplant ptC. Necrosis + INIB kidney, adrenal;

vasc endothelium, CNS DzD. Subclinical INIB in enterocytesE. Few renal transplant pt ;

++ SIV/suppressed macaquesF. Immunodef rats: Harderian

parotid lung lesions +INIB


COD /CCOD ANTImicrobial

• Streptozotocin STZ

• Diabetogenic• Targets islet beta cells• ALSO Nephrotoxic Hepatotoxic Strain/sex variations

63654



133

COD/CCOD PDX tumor?

• Primary tumor• ‘Excellent take’• PDX spleen

134

PDX recipient ‐ Excellent ‘take’?

• Human carcinoma

• PDX orthotopic + skin excellent take + splenomegaly + Leukemia???

• Telomerase confirms human or ???

• EBNA ++

• IHC ‐ hu /mouse cd45 also useful

BEWARE of EBV Lymphoma in NSG (PDX or humanized mice)Dieter, et al. (2017). "Patient‐derived xenografts of gastrointestinal cancers

are susceptible to rapid and delayed B‐lymphoproliferation." Int J Cancer 140(6): 1356‐1363.

Ma, et al. (2016). "PD‐1/CTLA‐4 Blockade Inhibits Epstein‐Barr Virus‐Induced Lymphoma Growth in a Cord Blood Humanized‐Mouse Model." PLoS Pathog 12(5): e1005642.

Lee, et al. (2015). "Effects of lymphocyte profile on development of EBV‐induced lymphoma subtypes in humanized mice." Proc Natl Acad Sci U S A 112(42): 13081‐13086.

Wetterauer, et al. (2015). "Early development of human lymphomas in a prostate cancer xenograft program using triple knock‐out immunocompromised mice." Prostate 75(6): 585‐592.

136

Beware of Biological MaterialsRecent (also historical) contaminants

Agents Some Phenotypes in immunodeficientMycoplasma sp. Respiratory dz + sheddingLDV Immunomodulate LE’s + sheddingHuman herpesviruses BIOHAZARD + sheddingHIV BIOHAZARD + sheddingHepatitis B BIOHAZARD + sheddingEBV BIOHAZARD + shedding, Lymphoproliferative DzParvoviruses Immunomodulate, Tumor growth ? + sheddingPolyoma virus Immunomodulate, Tumors + sheddingLCMV Immunomodulate, BIOHAZARD + sheddingMouse adenovirus Immunomodulate – INIB? Kid, Gut + sheddingMouse hepatitis virus Wasting + enterohepatic DZ + sheddingmCMV Death – hepatic etc necrosis + sheddingEctromelia virus Death & sheddingPneumonia virus of mice (PVM) Respiratory DZ + sheddingMurine norovirus Enteritis encephalitis n some + sheddingSendai virus Wasting + respiratory Dz + sheddingE. cuniculi Kid + CNS ? + shedding

• from CBW RADIL & CC CRL –AALAS 2010

SFB • Segmented Filamentous Bacteria

• Good for our mice?

• Th17 etc development…

• Our friends ?

• Florid growth suggests immunoweird +/‐ ill…

• Competent mice typically clear to very low levels by ~ 1mo With IgA production

COD/CCOD NOT

137 138

Segmented Filamentous Bacterium (SFB) of the Small Bowel of mammals and birds • Described by Savage, Dubos etc pre 1970’s

‘Savagella’ spp ?? [Arthromitus] Gram Positive, Spore former Attach to brush border

• Difficult to cultivate/culture not in Schaedler’s flora Grown on cells + complex media

Schnupf & al 2015

• Absent from common J strains – Ivanov & al. 2009. Induction of intestinal Th17

cells by segmented filamentous bacteria. Cell.



139

Robert Burns 1759-1796

To a Mouse c1785

The best-laid schemes o' mice an 'men

Gang aft agley,

An'lea'e us nought but grief an' pain,

For promis'd joy!

140

To a Mouse 2018

The phenotype/outcome we expect

Often (usually?) is not

The phenotype/outcome we get …

141141

Mighty Mouse

Also

• Don’t blame the pathologist when its not the answer you were aiming for ….

142

143

Thanks!!

Mice!

Hosts!

Sponsors!

Audience!

That’s all folks!



Documents

Comparative Histopathology (Mouse vs Human)mcp.bs.jhmi.edu/sites/default/files/18 BSTP BRayton ComPAth +COD 1 15.pdf · stomach) Ward et al. 2001. Hyalinosis andYm1/Ym2 gene expression