Mouse Pathology Spontaneous Phenotypes and Diseases (NON

2017 Brayton MICE NON infectious 32p

OPC Disclosures

Mouse Pathology No financial disclosures that I know of… Spontaneous Phenotypes All opinions expressed and implied in and Diseases (NON infectious) this presentation are solely those of Dr. Brayton. The content of the presentation does Cory Brayton, DVM, DipACLAM, DipACVP not represent or reflect the views of Associate Professor, Molecular and Comparative Pathobiology Director, Phenotyping Core Johns Hopkins University or Johns Johns Hopkins University, School of Medicine Hopkins Health system. Baltimore, MD 21205 [email protected] http://www.hopkinsmedicine.org/mcp/PHENOCORE/index.html 3

General References Resources online 1. ACLAM Blue books https://www.elsevier.com/books/book‐series/american‐college‐of‐ laboratory‐animal‐medicine 1. Brayton: Spontaneous diseases in commonly used mouse strains and stocks (2013) http://phenome.jax.org/projects/Brayton1 – The Mouse in Biomedical Research. Fox, Barthold, Davisson et al. Vol I‐IV 2006 – Lab manual at http://www.hopkinsmedicine.org/mcp/PHENOCORE/courseCURRENT.html – Laboratory Animal Medicine Fox et al 2015 2. DORA Diseases of Research Animals http://dora.missouri.edu/ 2. Barthold S, Griffey S, Percy D. 2016. Pathology of Laboratory Rodents and Rabbits, Wiley‐ Blackwell. 3. 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 3. Brayton C. 2006. Spontaneous Diseases in Commonly Used Mouse Strains The Mouse in http://store.cldavis.org/ Biomedical Research. Fox, Barthold, Davisson et al. New York, Elsevier (Academic Press). II. Diseases: 623‐717. outline at http://phenome.jax.org/projects/Brayton1 4. MGI Mouse Genome Informatics SEARCH GENES etc info http://www.informatics.jax.org/ 4. Brayton C, Treuting P, Ward J. 2012. Pathobiology of aging mice and GEM: background – IMSR ‐ FIND MICE AND ES CELL LINES http://www.findmice.org/index.jsp strains and experimental design. Vet Pathol. 49(1):85‐105. – Mouse (rat) and Gene NOMENCLATURE & apply for a laboratory code 5. Treuting P and Dintzis S, Eds. 2012. Comparative Anatomy and Histology: A Mouse and http://www.informatics.jax.org/mgihome/nomen/index.shtml Human Atlas. Elsevier (Academic Press): London. pp. 361‐381. – MPD‐ Mouse Phenome Database mouse data and protocols http://phenome.jax.org/ new edition in press 2017 –with RATS – MTB Database & tumor frequency grid http://tumor.informatics.jax.org/mtbwi/index.do Primary Journals 5. Noah’s Arkive (now at CLDavis) http://noahsarkive.cldavis.org/ 1. Comparative medicine / JAALAS (Search Comp Med, JAALAS) 6. NTP NON neoplastic lesion atlas http://ntp.niehs.nih.gov/nnl/ 2. Journal of Comparative Pathology (search J Comp Pathol) 7. Pathbase: European mutant mouse pathology database http://www.pathbase.net/ 3. [Laboratory animals (Sage); Lab An (NY) (Nature.com)] 8. RENI Tissue trimming guide + INHAND terminology and diagnostic criteria 4. Toxicologic Pathology (search toxicol pathol) http://www.goreni.org/ 5. Veterinary Pathology (search vet pathol) 9. VSPO JPC https://www.askjpc.org/vspo/

Etc. spontaneous conditions in Some Phenotypes (& Pathology) of recent literature: PubMed SEARCH ‘Normal’ mice (short list)

 vet pathol [jo] YEAR mice  Comp med [jo] YEAR mice  C57BL/6? Microphthalmia, hydrocephalus, ulcerative dermatitis, osteoporosis, presbyacusis, AMP, amyloidosis,  vet pathol [jo] YEAR rats  Comp med [jo] YEAR rats lymphoma, histiocytic sarcoma .…  129? Teratomas (Ter), lung tumors, AMP, hyalinosis, acallosity, megaesophagus, otitis, lymphoma …  toxicol pathol [jo] YEAR mice jaalas [jo] YEAR mice  FVB? rd1, seizures, lung tumors, AMP, mammary/ pituitary dz ….  toxicol pathol [jo] YEAR rats  jaalas [jo] YEAR rats

 j comp path [jo] YEAR mice  lab anim [jo] YEAR mice  DEAF‐ C57BL/6, BALB, DBA, etc  BLIND dt rd1 ‐ C3H, CBA, SJL, SWR, FVB  j comp path [jo] YEAR rats  lab anim [jo] YEAR rats  C5 deficient 9

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Phenotypes to expect When to expect them Discussion Plan

Malocclusion Younger mice Important Phenotypes Hydrocephalus (By weaning) 1. Sexual dimorphisms. Sexual dimorphisms Blind / Deaf 2. 2 killers to identify at/by weaning. SKIN/HAIR: Barbering / dermatitis Various ages 3. 3 Common clinical complaints. Imperforate vagina MUS  Hydronephrosis 4. 4 Sick old mouse phenotypes. Vestibular syndromes 5. Top 5 tumors Acidophilic Macrophage pneumonia AIM: DON’T LET THESE MESS UP YOUR RESEARCH Amyloidosis & glomerulonephritis Older mice Arteritis 6. A few more fun findings …. Tumors13 14

1. Some Sexual Dimorphisms SEX MATTERS  Prevalence of sexual dimorphism in mammalian Size  growth curves = NICE DATA ! phenotypic traits. . Are the GEM really smaller? Slower growing ? – Nat Commun. 2017 Jun 26;8:15475. Anogenital distance –tough in younger https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490203/ . In utero influences – How many authors does it take to convince a scientist?

Nipples –not in males  Karp et al: Mason J, Beaudet AL, Benjamini Y, Bower L, Braun RE, Brown SDM, Chesler EJ, Dickinson ME, Flenniken AM, Fuchs H, Angelis MH, Gao X, Guo S, Greenaway S, Heller R, Herault Y, Justice MJ, Kurbatova N, Lelliott CJ, Lloyd KCK, Mallon AM, Mank JE, Masuya H, Salivary glands – bigger in males McKerlie C, Meehan TF, Mott RF, Murray SA, Parkinson H, Ramirez‐Solis R, Santos L, Seavitt JR, Smedley D, Sorg T, Speak AO, Steel KP, Svenson KL; International Mouse Phenotyping Consortium, Wakana S, West D, Wells S, Westerberg H, Yaacoby S, White JK. +159 . Acidophilic tubules Collaborators: bata Y, Suzuki T, Tamura M, Kaneda H, Furuse T, 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, Kidneys – bigger in females ? Males ? Newbigging S, Morikawa L, Law N, Shang X, Feugas P, Wang Y, Eskandarian M, Zhu Y, Nutter LMJ, Penton V,P, Laurin Clarke S, Lan Q, Sohel K, Miller D, Clark G, Hunter J, Cabezas J, Bubshait M, Carroll T, Tondat S, MacMaster S, Pereira M, Gertsenstein M, Danisment O, 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, Radage A, Maritati M, Mianne J, Gardiner . Cuboidal parietal epithelium –male (>> female) 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, Kipp C, 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 DT, Vancollie VE, Pearson SA, Gates AS, Sanderson M, Shannon C, Anthony LFE, Sumowski MT, McLaren RSB, Swiatkowska A, Adrenal glands – bigger in females 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 Marchand E, El Amri A, Vasseur L, Aguilar‐Pimentel A, Becker L, Treise I, Moreth K, Stoeger T, Amarie OV, Neff F, Wurst W, Bekeredjian R, Ollert M, Klopstock T, Calzada‐Wack J, Marschall S, Brommage R, . X zone (vacuolar change in females) 16 Steinkamp R, Lengger C, Östereicher MA, Maier H, Stoeger C, Leuchtenberger S, Yildrim A, Garrett L, Hölter SM, Zimprich A, Seisenberger C, 15 Bürger A, Graw J, Eickelberg O, Zimmer A, Wolf E, Busch DH, Klingenspor M, Schmidt‐Weber C, Gailus‐Durner V, Beckers J, Rathkolb B, Rozman J.

Sex and SIZE 1. Some Sexual Dimorphisms (+ Genetic Background) ♂ If they’re normal, SIZE (body weight) what’s the big deal? Cheapest Data you can get?  M > F – B6D2F1 ~ Hi? Are there sexual dimorphisms in the data, or – C3H/HeJ ~ lo research endpoints ?  Jax Phenome Database   ANALYZE Male & Female data separately data by strain sex age protocol – Jax Pheno1 ♀ ALSO CONSIDER: NOT having these may be – n = 5‐15 an important phenotype – 6% fat diet NIH 316 Purina 5K52 17 18

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Sexing pups Salivary Glands, associated Nodes Nice pictures at http://www.thefunmouse.com/info/sexing.cfm Which is Male ? A A or B ?

1. Submandibular 2. Sublingual B 3. Parotid  (exorbital lacrimal) 4. Lymph nodes

Nipples may be evident by about 6do 21

Tissues? Tissue? Which is Male? Which is more likely to be male? AB AB

22 23

Tissue? Which is Female ? 1. Sexual Dimorphisms

ABFEMALE  Size  Growth Curves = NICE DATA !  Salivary glands, Kidneys, Adrenal glands etc  Many More

CONCLUSIONS:  EXPECT THE EXPECTED – consider the unexpected  ANALYZE Male & Female data separately 24 25 Missing Dimorphisms may be a phenotype [email protected] 2017 Page 3 of 32 2017 Brayton MICE NON infectious 32p

2. 2 Young Mouse Phenotypes Hydrocephalus to identify by / at weaning: Especially C57BL 1. Hydrocephalus . Later onset not evident at weaning  E.g. sporadic in MRL etc. 2. Malocclusion / Incisor overgrowth . Cull or trim . Breed?? 3. Microphthalmia . Expect this in C57BL –but it won’t kill them

26 . Especially? Right eye in females 27

Malocclusion , incisor overgrowth 2. 2 Young Mouse Phenotypes  Secondary Phenotypes to identify by / at weaning: 1. Hydrocephalus 2. Malocclusion / Incisor overgrowth 3. Microphthalmia etc dysmorphisms

6g –6 weeks old CONCLUSIONS: ‘Developmental delay’  EXPECT THE EXPECTED – consider the unexpected – important phenotype? or just not eating (inanition)?  DON’T let these kill your mice !  Examine your mice ! 28Early Death 29 i i d i ih h

Pup assessments

p0 Eyes closed Open ~p14 Ears closed Open ~p3‐5 MILK spot By 6hr… Not evident (externally) by approx p6 NO FUR Fuzz starts ~p2‐6 NO incisors Erupting ~p11 Nibbling solid food ~p12 NO nipples females ~p9 Males should NOT have them

• RECORD/REPORT:  Weight  Milk spot (food in stomach in older pups)

 When little or no milk is present by 6 hours after birth, it is almost certainly the case that something is wrong with either the pup or the mother. ‐Silver  Eyes open/closed/color  Ears  Teeth –check for cleft lip/palate in neonates)  Fur, nipples, sex by anogenital distance

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3. 3 Common 1st Clinical Complaint: Skin Clinical Complaints 1) Skin Disease Alopecia 1) Barbering more common 2) Ulcerative Dermatitis more serious – Social phenomenon F > M 3) Flakey skin ‐ in immunodeficient – discussed later 4) Tumors discussed later – Vibrissae are important in 5) Mites discussed frequently some behavior tests … 2) Abdominal distention 1) Pregnancy  2) Hydrometra mucometra pyometra 3) Hydronephrosis (MUS) 4) Neoplasia – effusion, organomegaly 5) Effusion ‐ Neoplasia ? Heart or kidney disease?

323) Neurologic signs 33

Skin Alopecia SKIN: ulcerative dermatitis Barbering Mouse Ulcerative – Strain/sex 129S1 ♂ NMRI ♂ C57Bl/6 Dermatitis (MUD) specific patterns . Interferes with a lot of research – Nibbled . Secondary phenotypes: Leukocytosis . Not plucked – Lymphadenomegaly, ouch! Splenomegaly C57Bl/6 ♀ NMRI ♀ A/J ♂ – Vibrissae are Sick mice important in  With Amyloidosis?  With GN some behavior glomerulonephritis? tests … C57BL, Swiss > others?? 35 Kaluef34 f et al. 2006. ♂

MUD mouse ulcerative dermatitis Ringtail   Strains B6 Swiss etc  Age Age Younger 12‐24m  Sites Diet Restriction Ad lib  Strains? Female +/‐  Causes? Drug Rx 0‐ min effect Toenail trim Dorsal neck Flank  Lo humidity ?  Cornification  Cause/s: undetermined –multifactorial disorder?  Cure? Pedicure (toenail trim) helps many

Chu, D. K., et al. (2016). "Systematic Literature Review of Risk Factors and C. Recordati et al. Vet Pathol 2014;52:700-711 Copyright © by American College of Veterinary Pathologists Treatments for Ulcerative Dermatitis in C57BL/6 Mice." Comp Med 66(2): 89.

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Cause ? Part of the phenotype?

 Preweaning  Tail, digits, and less frequently limbs  Various strains  Low humidity? Etc environmental  disorder of cornification ? persistent keratin ring ? strangulating exremity  edema, thrombosis, hemorrhage, necrosis, …auto‐amputation C. Recordati et al. Vet Pathol 2014;52:700-711 C. Recordati et al. Vet Pathol 2014;52:700-711 Copyright © by American College of Veterinary Pathologists Copyright © by American College of Veterinary Pathologists

GEM Model Abscesses

Transgenic Mice Often one end or the other Expressing a Mutant Form of Loricrin Reveal the Molecular Basis of the Skin Diseases, Vohwinkel Syndrome and Progressive Symmetric Erythrokeratoderma  Infectious dz lecture Yasushi Suga et al. J Cell Biol 2000;151:401-412 © 2000 The Rockefeller University Press 43

Facial Facial Abscesses ABSCESSES!  Lawson 2010 Folliculitis  Furunculosis ?  mandibulofacial and maxillofacial abscesses

Myd88 deficient

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Foreign body 2nd Clinical Complaint periodontitis Abdominal distention  Common finding in ‘Pregnant x weeks… months ’ aging mice of any? Don’t ignore – NOT likely to have pups…. Strain? Mouse gestation ~ 19‐21 d…..

 Not so frequently associated with abscesses?

Lawson 2010 47

Hydronephrosis Hydrometra, mucometra

Progressive abdominal distention – weeks … – Imperforate vagina – Vaginal septa – Esp BALB/c, B6? – Note perineal bulge  In Males ‐ Probably / Usually Urinary obstruction – Usually infertile – Has been called mouse urologic syndrome (MUS) – Pyometra –  Role of Genetic Background ?? Diet ?? Caging ?? discussed later  Polycystic kidneys are polycystic – 48 – Multiple cysts, genetic 49

WYD? Whats your diagnosis (WYD)? A. Pregnant A. Pregnant B. Obese B. Obese C. Ascites/anasarca C. Ascites/anasarca D. Hydronephrosis D. Hydronephrosis E. Hydrometra  http://www.ahwla.org.uk/ E. Hydrometra  Cause/s?  Cause/s? A. Tumor effusion A. Tumor effusion B. Antibody production B. Antibody production C. Cardiomyopathy C. Cardiomyopathy D. Renal disease D. Renal disease E. Imperforate vagina E. Imperforate vagina

50 51 Sussman & al 1999

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3rd Clinical Complaint Whats your diagnosis (WYD)? ‘Neurologic’ A. Pregnant 1. Paralysis, posterior paresis B. Obese a) Neoplasia – discussed later C. Ascites/anasarca b) Infectious demyelination –MHV, TMEV – discussed later D. Hydronephrosis c) Autoimmune demyelination – Induced –EAE etc E. Hydrometra d) Trauma, induced, muscular dystrophy? other 2. Spinning, Rolling ‐ Dizzy, Vestibular a) Otitis – infectious – discussed later b) Arteritis – discussed later c) Infarct ~ stroke ? d) Trauma, induced, other

3. Seizures (sudden death) a) FVB/N, DBA/2 other genetically susceptible b) Other ‐ Induced? Chemical ? Genetic ? 52 https://www.sciencenews.org/ 9/9/13 53

'Neurologic' #2: Rolling, 'Neurologic‘ #1: Posterior Paresis Spinning, Head tilt (vestibular) Important phenotype? Dizzy mice – GEM Model 1. Neoplasia of vestibular syndrome? 2. Infectious demyelination . MHV, TMEV, LDV Consider: 3. Autoimmune demyelination 1. Otitis? . Induced –EAEetc 2. Arteritis ? 4. Other Induced/experimental? 3. Tumors? . Iatrogenic? Trauma? 4. Other? . Muscular dystrophy? 54 . other ? 55

Dumb mice? Malacia, necrosis compatible or Dizzy ? with ischemia/infarct (Stroke) Vestibular Signs  Southard & al 2007, 2010

 Acute onset spinning (vestibular signs)   death Rolling, turning Head Tilt

 >12 mice of 2 Swiss stocks; 1 source –local 56 57

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3. 3 Common 'Neurologic' #3: Seizures Clinical Complaints Important phenotype? 1. Skin Disease In what strains do you expect seizures ?? 2. Abdominal distention . FVB/N 3. Neurologic signs . DBA/2 CONCLUSIONS: Consider  EXPECT THE EXPECTED – consider the . Tumors unexpected… . Induced ‐ Kainic acid etc, surgical, IC inoculation  Examine your mice…  Consider pathology to assess further… 58 59 Don’t let these mess up your research.

4. 4 Sick Old Mouse 4.1 Amyloidosis Phenotypes (Phenotype: wasting, 'premature aging' ) 1. Amyloidosis – multisystem Rule out glomerulonephritis 2. Arteritis – multisystem 3. Hyalinosis & Acidophilic macrophage pneumonia 4. Neoplasia Sporadic – common on different genetic backgrounds Histopathology to diagnose

 MANY other conditions – but not so likely to contribute to morbidity mortality, e.g. Angiectasis, melanosis, mild mineralization, inflammatory infiltrates, pancreatic … Intestine (ileum) first & worst . Usually earlier & more severe than kidney, etc 60 61

Amyloidosis, kidney Mouse Amyloidosis (pink stuff in glomeruli) Phenotypes

 Gut – Intestinal amyloid  malabsorption  Kidney – Glomerular amyloid  protein loss  Liver –Hepatic amyloid size, function . Adrenal Weight loss & Wasting . Salivary glands ‘Premature Aging’ phenotype . Tongue e.g. in SAMP Senescence Accelerated Mice . Heart . Stomach . Testes 62 63

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Mouse Amyloid Apoa2 gene/locus allele Strains 2 types AKR, BTBR, C57BL/6J, C57BL/10, DBA/2 ApoA2a WERE COMMON complicators of chronic SAM‐P3, SAM‐P8, SAM‐R2 129S1, BALB/CJ, BALB/cByJ, C3H/, CBA, FVB/NJ, MRL, NZW etc ApoA2b studies SAM‐P6, SAM‐R1 – less ‘clean’ conditions c A/J, SJL/J etc ApoA2 SAM‐P1, SAM‐P2, SAM‐P7, SAM‐P9, 1. Reactive, Secondary, AA –SAA ApoA2d PERA, WSB . SAA serum amyloid A component ApoA2e SPRET Acute phase response protein  c allele  EARLY ASSAM onset in liver . chronic immune stimulation –GI, kidney, spleen spleen 2. Senile, ASSAM amyloid – Apoa2c >> Apoa2a . ‘aging phenotype’ . Apolipoprotein A2 (part of HDL )  a allele ‐ later onset in gut, lung, tongue etc. . Alleles a,b,c,d,e  b allele –QTL associated with higher HDL level 64 65 . Summarized from Higuchi & al 1991; Wang & al 2004

Pink Stuff in Glomeruli More pink stuff in glomeruli  Amyloid is Congo Red POSITIVE / PAS negative A. H&E amphophilic and amorphous material in  PAS POSITIVE interstitium and – Glomerulonephritis or glomerular hyalinosis or ??? glomerulus. H&E PAS B. C, D Congo Red – Difference between B, D E. PAS F. Trichrome  Diagnosis?

Hyaline glomerulopathy CR 8µm

 Enlarged glomeruli CR  5µm PAS positive  Massons trichrome light blue‐purple PAS Tri  Congo Red negative

Amyloidosis Hyaline glomerulopathy – Intracapillary hyaline • Interstitial + glomerular •Expanded Bowman space thrombi •Hypercellular mesangium • Congophilia improved by

Hoane et al thicker. sections, glass coverslip •Mesangial /subendothelial deposition Hoane et al . Copyright © 2016 Society of Toxicologic Pathology Copyright © 2016 Society of Toxicologic Pathology •‘Hyaline thrombi’ (*)

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Pink Stuff in Kidney glomeruli Glomeruli  Technique/s? Renal Glomerular Amyloid – UV evaluation for vs autofluorescence Renal Hyaline Glomerulopathy  Diagnosis/es? A/B ? C/D ? – Amyloidosis B6C3F1 Mice from NTP Chronic – Hyaline Studies glomerulopathy

Pink Stuff in Glomeruli Beware of GEM…

8mo, female, B6C3Fe-a/a-Cola2oim/oim 72 8 mo, female, B6C3Fe-a/a-Cola2oim/oim 73 Fibrocollagen glomerulopathy

Old Mouse Kidneys 4.2 Arteritis, periarteritis pink stuff in glomeruli  Often incidental, not associated with clinical signs, in chronic GN Membranoproliferative PAS+ thick BM studies on various strains  with glomerulosclerosis Small trichrome ++ Also implicated in Vestibular syndrome, or death (with severe cardiac involvement) Amyloidosis Glomeruli CONGO+ (Tri) +/‐ interstitial Heart Head Hyaline glomerulopathy PAS+ (Tri)

Fibrocollagen glomerulopathy TRICHROME++ GM?

74 75

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4.3. Acidophilic Macrophage Acidophilic Macrophage or Crystalline ‘Pneumonia’ Acidophilic Crystalline ‘Pneumonia’ Pretty histology Spectrum of clinical Phenotypes

No clinical disease TO Respiratory distress, death

Spectrum of Anatomic Pathology Phenotypes

Scattered Acidophilic Pulmonary consolidation by Macrophages or TO acidophilic macrophages & Crystals crystals  Associations with Pneumocystis etc. agents? –In immunocompromised / immunoweird mice especially? On B6 or 129 background ? 76 77

Thoolen, Maronpot et al. 2010. Proliferative and Nonproliferative Lesions of the Rat ‘Hyalinosis’ in other tissues and Mouse Hepatobiliary System. Tox Path. INHAND Nomenclature Fig 25. Rat liver. Pigment in hypertrophic Pretty histo – nose, stomach, gall bladder hepatocytes consistent with bile. Cholestasis. Crystals in marrow spleen – macrophages, PMN Fig 26. Mouse liver. Hyalinosis of hyperplastic bile ducts with crystal formation and peribiliary inflammatory cell infiltrate. Fig 27. Mouse liver. Hyalinosis of hyperplastic bile ducts with crystals and peribiliary inflammatory cell infiltrate. Higher magnification of Figure 26. Fig 28. Mouse liver. Hyalinosis of hyperplastic bile ducts with crystal formation. Fig 29. Mouse liver. Numerous intracytoplasmic hyaline bodies in an hepatocellular adenoma. Fig 30. Mouse liver. Intranuclear inclusion 78 79body, cytoplasmic invagination. Copyright © by Society of Toxicologic Pathology

Chil3 (Ym1), YM2 chitinase like proteins Acidophilic Macrophage Pneumonia in Hyalinosis & Acidophilic Macrophage Pneumonia Eosinophilic Crystalline 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… . Lung ‐‐ Ym1‐expressing cells are alveolar macrophages, Ym1 localized in RER. . Spleen ‐‐Ym1‐expressing cells in red pulp identified as immature neutrophils. 80 . Marrow ‐‐ immature neutrophils  lose immunoreactivity with maturation. 1990 (Murray & Luz)

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Eosinophils, granules 4. 4 Sick Old Mouse Charcot Leyden Crystals Phenotypes

1. Amyloidosis – multisystem Rule out glomerulonephritis etc… 2. Arteritis – multisystem 3. Hyalinosis & Acidophilic macrophage pneumonia 4. Neoplasia – discussed Next CONCLUSIONS  EXPECT THE EXPECTED –consider the UNexpected …  PATHOLOGY to diagnose…  Don’t let expected conditions mess up your research! 1990 (Murray & Luz) 83

More Old Mouse lesions WYD? Incidentalomas Nose Mouse. – Submucosal eosinophilic material non Amyloid ‘Amyloid’ in – Hyalinosis (YM1,2 Chi3 like 3) submucosa of nasal septum. Teeth NOT amyloid – MOLARS Periodontal inflammation (hairs), alveolar bone remodeling hypercementosis – Congo red NEGATIVE – INCISORS Dysplasia (denticles) – PAS Positive

Eosinophilic Substance Deposition in Mouse Nasal Septum Molars  Sex Difference M – M>F  ‘No Increase in Seniles’ – increases w age – males plateaus by 58 wo F – femalesby 34 wo.  Congo Red negative  PAS Positive Doi et al. Toxicol Pathol 2010;38:631-636 Copyright © by Society of Toxicologic Pathology 87

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Incisors Incisors

 Dysplasia  Incidental finding?  Tumor?  CoD?

88 89

More (old & not so old) mouse lesions Li et al. 2016. MG

CONSIDER THE STRAIN DIAGNOSES?  Cardiovascular BALB/c, C3H, DBA, A/J – Fibrosis, mineralization  Soft tissue mineralization BALB/c, C3H, DBA, A/J, KK…. – (Hypertrophy?)  Muscle A/J, SJL/J Susceptible strains?  Etc… – C3H, DBA, BALB/c, KK etc Implicated genes? – Abcc6 (Dyscalc1) Relevant human condition? – pseudoxanthoma elasticum (PXE)

Li, Q., et al. (2016). "Mouse genome‐wide association study identifies polymorphisms on chromosomes 4, 11, and 90 15 for age‐related cardiac fibrosis." Mamm Genome.

HEART Glass et al. 2013. Comp Med. DIAGNOSES?  Diagnosis/es? – Epicardial mineralization – Epicardial mineralization akaCardiac calcinosis – DCC dystrophic cardiac calcinosis  Variation by source Likely strains? /strain: – BALB/c, A/J, KK etc – Which is BALB/cByJ ? A or B? Special Stain ? – Which is BALB/c(AnTac) ? – Von Kossa A or B?

"Spontaneous cardiac calcinosis in BALB/cByJ mice." Comp Med 63(1): 29‐37.

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DiPaolo et al. 1964 Glass, A. M., et al. (2013). "Spontaneous cardiac calcinosis in BALB/cByJ mice." Comparative Medicine 63(1): 29‐37. DiPaolo, * Leonell C. STRONG & Moore. 1964. CALCAREOUS PERICARDITIS IN  BALB/cByJ lesions MICE OF SEVERAL GENETICALLY contain calcium, RELATED STRAINS. collagen, and inflammatory cells DBAxBagg  A A Alizarin red S DBAxA  C DBA Swiss Orange affected Strain n %  Brown = mineral DBA/2 60 74 81 C/St 39 56 70 B,C Masson C3H/St 19 56 34 trichrome DBA/St 6 26 22 CHI/St 4 29 14 Blue = collagen ICR/Ha 10 112 9 Those who do not learn from history and pathology D inflammation should be condemned to rejection or retraction ….

KK mice A/J Dysferlinopathy Dysfim  Diagnosis/es? Early changes, degeneration regeneration – Soft tissue mineralization  Human Relevance? – Pseudoxanthoma elasticum?  Relevant genes? – Abcc6 ? (Dyscalc1)

Fibroosseous lesions

 Diagnosis/es? – Bone, marrow, hyperplasia, fibrovascular, fibro osseous; – Loss of hematopoietic tissue  Older mice  Females (>males)  Estrogens  Predisposed strains – KK > 129S1/SvImJ, C3H/HeJ, and DBA/2J, etc  Implicated genes?

– Abcc6 ? etc http://noahsarkive.cldavis.org/cgi-bin/101 show_image_info_detail.cgi?image=F08422

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Mouse Diagnosis/es?  Diagnosis/es ?  Megaesophagus, thoracic  Likely Strain?  129, related mice .

 Strain ?  Other causes of  Mutation ? urolithiasis ?

Livrozet M, Vandermeersch S, Mesnard L, Thioulouse E, Jaubert J, et al. (2014) 102 http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0102700

Figure 2. 129S2/SvPasCrl crystalluria and aminoaciduria are similar to those of human patients with cystinuria. WYD?

 Strain?  Cause?  Age?  Significance ?

Livrozet M, Vandermeersch S, Mesnard L, Thioulouse E, Jaubert J, et al. (2014) An Animal Model of Type A Cystinuria Due to p75 neurotrophin receptor Spontaneous Mutation in 129S2/SvPasCrl Mice. PLoS ONE 9(7): e102700. doi:10.1371/journal.pone.0102700 PAS http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0102700 (p75NTR) IHC

WYD More (old & not so old) mouse lesions CONSIDER the interventions

 Irradiation  Antimicobials

107 110

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NODscid Mortality post irradiation CD‐1 Not just an incidental finding A. Chronic keratitis, keratinization and necrosis of corneal epithelium, infiltration by neutrophils. B. Severe chronic keratitis; thickening and keratinization of the epithelium, and fibrosis and neovascularization of the stroma Larsen & al. 2006. Mukaratirwa et al. Toxicol Pathol 2014;43:530-535 Copyright © by Society of Toxicologic Pathology

Appreciate the ameloblasts… C57BL/6 NOS

 Which is normal?  A  Diagnosis/es?  Enamel hypomineralization  Causes?  amoxicillin/clavulanic acid Rx  Human relevance?  Amoxicillin Rx in early childhood may be associated with enamel

Mihalaş et al. hypomineralization Mihalaş etCopyright al. © by Society of Toxicologic Pathology Copyright © 2015 by Society of Toxicologic Pathology http://journals.sagepub.com/doi/abs/10.1177/0192623315610822 Copyright ©Eugeniu 2015 by Mihala Societyş et of l. ToxicolToxicologic Pathol Pathology 2015;44:61-70 http://journals.sagepub.com/doi/abs/10.1177/0192623315610822

Incisor  (A) 100 & (B) 150 mg/kg BW of clavamox  Cyst‐like lesions under transitional ameloblasts  Amoxicillin in childhood associated with enamel hypomineralization  In B6: dysfunction in maturation and transitional ameloblasts , resulting in hypomineralized Altholtz, L. Y., et al. (2006). "Dose-dependant hypothyroidism in mice induced by enamel commercial trimethoprim-sulfamethoxazole rodent feed." Comp Med 56(5): 395-

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Mouse Cancer Quiz 5. 5 Mouse Tumors Tumor Sex Strain predisposition 1. Thymic Lymphoma a) A Hematopoietic AKR, SJL/J, NODscid F>M 2. B cell Lymphoma b) AKR, NODscid, C58 Lymphoma, HS (C57BL, etc) 3. Plasma cell tumors (myeloma) Lung A (FVB, 129, etc) (Harderian, myoepithelioma, c) BALB/c Mammary F>>M C3H (BALB etc) Rhabdomyosarcoma etc ) d) C3H Liver M>>F C3H, CBA, (B6C3 …) 4. Lung tumors e) C57BL/6 Pituitary F > M 5. Mammary tumors 6. Mammary Hyperplasia, f) FVB/N  What strains are famous for what tumor? EMT with pituitary tumors g) SJL/J . Good or Bad for your model ?  7. Liver tumors WHAT SHOULD YOU EXPECT IN YOUR MICE ? 117  Is your favorite strain tumor resistant?  8. Cancer resistant 118

Rodent Cancer Classification ! Hematopoietic Tumors Mouse V Human MMHCC – http://emice.nci.nih.gov/  Lymphomas  Leukemias – mouse models of human cancer consortium – Thymic L (younger) – ALL in children . Consensus papers hematopoietic liver mammary – Follicular B cell – AML in adult prostate intestine ….  Histiocytic sarcoma  Lymphomas . GEM oriented  Retroviruses as genetic – Diffuse large B cell INHAND http://www.toxpath.org/nomen/ engineers  FCC lymphomas Common features – International Harmonization of Nomenclature • and Diagnostic Criteria in Toxicologic Point mutations Pathology • Activated Oncogenes • Inactivated Tumor suppressors 119 . Rat is complete, mouse is coming 120

Mouse Hematopoietic Tumors Mouse Hematopoietic Tumors Spontaneous Spontaneous #1 Distribution T‐cell lymphoma B‐cell lymphoma Histiocytic sarcoma Myeloid leukemia Thymic lymphoma (T lymphoplastic Systemic Common Rare Rare Rare (generalized) Lymphoma) Thymus Primarily Rare Rare Rare . B cell tumors are rare in thymus Common–follicular Occasionally –red pulp Spleen Rare Common or marginal zone origin Early onset often < 1y Peyer's patches Rare Common Occasional Rare . Thymus 1st  disseminates Mesenteric Lnodes Rare Common Occasional Rare Liver Rare Rare Common Rare .  Short studies … in Uterus Rare Rare Common Rare AKR, C58, scid, NODscid Peritoneum Rare Rare Common Rare Skin Rare Rare Occasional Rare Bone marrow Rare Rare Rare Occasional Kras activation IHC CD3 CD45r (B220) Pax5 CD68, F480 Endogenous retroviruses 121 From Table 1 - Ward 2006 Lymphomas and leukemias in mice. 122

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Mouse Hematopoietic Tumors Mouse Hematopoietic Tumors Spontaneous #1 Spontaneous #1  Clinical Signs: Histology  Gross Lesions:

 Overall ‘blue – Starry sky ?

 Blue cells +  tingible body macrophages’

123 123 124

WYD?

 Lymphoblastic Lymphoma

ATLS Figure 4. Electron micrographs of malignant lymphoblasts and cell lysis. Acute Tumor Lysis Syndrome Treuting & al 2010 Disseminated lymphoblastic lymphoma Mutator mouse model lung (94%), kidney (61%), liver (23%), brain (16%), and spleen (16%) of affected mice . Sporadic in other mice

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Figure 5. Detailed ultrastructure of pulmonary microthromboemboli. Mouse Hematopoietic Tumors Spontaneous #2

Follicular B cell lymphoma – Not exactly FCC ‐  Most common tumor or lymphoma in some strains  GALT, nodes, spleen etc  Later onset often > 1y  Expect it in control mice > 1yr

Mouse Hematopoietic Tumors Mouse Hematopoietic Tumors Spontaneous #2 Spontaneous #2  Clinical Findings: Lymph node  Gross Lesions: . Blue tumor . Monotonous population of similar cells with not much cytoplasm – Lymphoma.

. Leukemia ? 131 132 131

Mouse Hematopoietic Tumors Mouse Hematopoietic Tumors Spontaneous #3 Spontaneous #3

Histiocytic Sarcoma Histiocytic Sarcoma  Most common tumor in some strains – Organomegaly . Liver  Liver Uterus spleen nodes etc . Uterus  Later onset often > 1y . Invades anywhere  Expect it in control mice > 1yr – Pleomorphic ‐  Not such a blue tumor … sarcomatous  Not so round cells either … – Not so blue  (Diffuse large B cell lymphoma – Erythrophagocytosis can be quite pleomorphic too) . 134 133 Clker.com Can be prominent

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Mouse Hematopoietic Tumors 'Paralyzed mouse' Spontaneous #3 CONSIDER: – Neoplasia . Lymphoma . Histiocytic sarcoma . Sarcoma, poorly differentiated or osteosarcoma, esp in Trp53 mutant – Demyelination? . MHV, TMEV, induced – Trauma? Fracture etc  HS –not so blue – Very nasty too 135  PATHOLOGY – Very Pleomorphic here 136

Paralyzed mouse Tissues from 2 ‘paralyzed’ mice

 Not the desired  A  B. Neurologic phenotype…..

137

UTERUS WYD? mouse

 A  B.  Diagnosis/es? a) Endometrial adenocarcinoma b) Endometrial Stromal Sarcoma c) Granular Cell tumor d) Histiocytic Sarcoma e) Lymphoma  Other tissues affected?

Dixon et al. 2014 Copyri–ght Liver© by Society of Toxicologic Pathology https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253081/

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Mouse Hematopoietic Tumors 1. 8mo thymic T Lymphoblastic Spontaneous lymphoma 2. 6mo Lymphoblastic Lymphomas & histiocytic sarcoma B-cell lymphoma in mediastinum – Especially in old mice & susceptible strains 3. CD3+ T lymphoblastic T-cell lymphoma Leukemias not so common BUT 4. TDT+ cell nuclei in a T lymphoblastic advanced lymphoma or histiocytic lymphoma sarcoma likely to involve marrow, blood 5. TDT+ cell nuclei in a T lymphoblastic – Ulcerative dermatitis and nasty infections lymphoma 6. Follicular lymphoma are more common/likely causes of elevated in a Peyer’s patch of a 21-momouse WBC141 in mice. Rehg et al. Toxicol Pathol 2015;43:1025-1034 Copyright © by Society of Toxicologic Pathology

Hematolymphoid neoplasms MOUSE HEMATOLYMPHOID IHC from

• Rehg, et al. (2015). "Immunophenotype of Spontaneous Hematolymphoid Tumors Occurring in Young and Aging Female CD‐1 Mice." Toxicol Pathol 43(7): 1025‐1034. • Rehg, et al. (2012). "The utility of immunohistochemistry for the identification of hematopoietic and lymphoid cells in normal tissues and interpretation of proliferative and inflammatory lesions of mice and rats." Toxicologic Pathology 40(2): 345‐374. • Hao, et al. (2010). "The histopathologic and molecular basis for the diagnosis of histiocytic sarcoma and histiocyte‐associated lymphoma of mice." Vet Pathol 47(3): 434‐445.  <12mo: T&B lymphoblastic lymphomas • Ward & Rehg (2014). "Rodent immunohistochemistry:  >12mo lymphoblastic non‐lymphoblastic B‐cell lymphomas pitfalls and troubleshooting." Vet Pathol 51(1): 88‐101.  Renal hyaline droplets with a histiocytic‐associated large B‐ cell lymphoma (HA‐BCL) and a myeloid leukemia  Endogenous ecotropic mouse leukemia virus (MuLV) ALSO FYI NIEHS /NTP protocols: Rehg et al. Toxicol Pathol 2015;43:1025-1034 Copyright © by Society of Toxicologic Pathology https://www.niehs.nih.gov/research/resources/protocols/protocols‐immuno/index.cfm

Antigen Major cells expressing antigenHematolymphoid CELL SITE Antibody Source BCL6 Germinal center (GC) B‐cells (centroblasts and centrocytes), GC or post NUC RAB anti‐human Santa Cruz GC B‐cell neoplasms CD3 T‐cells and many T‐cell neoplasms MEM Goat anti‐mouse Santa Cruz CD4 FROZEN CD4+Tcells MEM CD5 T‐cells and many T‐cell lymphomas MEM Rat anti‐mouse BD CD8 FROZEN CD8+Tcells MEM CD43 Leukosialin ‐ Most/all hematolymphoid; Early B‐progenitors, T‐cells, myeloid cells, plasma cells, pro B‐lymphoblastic lymphoma, T‐cell MEM/surf Rat anti‐mouse BD lymphomas, myeloid leukemia, plasmacytomas; CD45 Common leukocyte antigen –all hematolymphoid except some plasma MEM paranuc CD45R/B220 Pan B: B‐cells, subsets of T‐cells, dendritic cells and NK cells Rat anti‐mouse BD CD138 Plasma cells (fibroblasts keratinocytes hepatocytes) MEM Rat anti‐ mouse BD F4/80 MAC in RED pulp, NOT white; Kupffer; round cells in HS, not fusiform MEM GATA1 Erythroid mega EO Baso Mast; Erythroid+ Mega Leukemia NUC (Transcription factor ) IBA‐1 Histiocytes/macrophages ANWHERE CYT RAB anti‐human Biocare Ionized calcium–binding adapter molecule 1 IFR4/MUM1 Normal and neoplastic plasma cells; post GC B‐cell lymphomas. Goat anti‐mouse Santa Cruz IgM Ig heavy chains, some immature B‐ cells; mature B‐cells; plasma cells CYT Rat anti‐mouse BD IgK Immunoglobulin kappa light chains, some immature B‐cells; mature B‐ Southern CYT Goat anti‐mouse cells; plasma cells Biotechnology Lysozyme Histiocytes/monocytes and myeloid cells (benign and neoplastic) CYT RAB anti‐human DAKO (LYZ) Better in round cells HS than Kupffer MAC‐2Macs in Red & white pulp most HS CYT nuc (MPO) Myelo peroxidase Myeloid cells benign and neoplastic Surf CYT RAB anti‐human DAKO PAX5 Paired box 5 B‐cells; B lymphomas; not expressed in plasma cells NUC Goat anti‐human Santa Cruz PNA Lectin germinal center GC surf; immature T‐cells of mice and human (FDC) SURF n/a Vector RUNX1/AML Myeloid cells, megakaryocytes, lymphoid; transcription differentiation NUC RAB anti‐human Active Motif TDT Terminal deoxyribonucleotidyl transferase; Immature thymic lymphoid NUC RAB anti‐human Supertechs cells, PRO B &PRE B lymphoblastic neoplasms VWF FVIII Megakaryocytes + ENDOTHELIAL CYT Rehg et al. Toxicol Pathol 2015;43:1025-1034 Copyright © by Society of Toxicologic Pathology YM1 (CHIL3) Neutrophils; Th2 activated macrophages + YM1 crystals CYT Goat anti‐mouse R&D

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Mouse Lung Tumors Mouse Lung Tumors Especially A strain mice Adenomas  Adenoma > Carcinoma

Small  Adenoma ‐ smaller Discrete – Patterns: Solid > papillary > mixed Compress  Carcinomas ‐ bigger – Papillary > mixed – May metastasize to liver  Wang & al. 2006.  Look like spontaneous  Nikitin, & al. (2004). but Kras+/‐ &TRP53 TG 147 148

Mouse Lung Tumors Lung Tumors Terminology Mouse V Human MMHCC: “Lung, adenoma or carcinoma”  Common in A >>  Most common . Withhold the term ‘Bronchioloalveolar’ . 129, FVB/N >> B6, DBA neoplasm? INHAND/Tox: A/B is still used  Peripheral  Most common CA death . ‘Alveolar/Bronchiolar’ – Descriptive, but may not be cell of . Bronchiolar v alveolar  NSCLC (80–85%) origin … DO NOT confuse with: v clara cells  Multiple –often – Adca increasing – Smoking carcinogens Human BAC = adenocarcinoma with pure  Adenoma > Carcinoma  SCLC (15–20%) bronchioloalveolar growth pattern, no stromal,  Kras activation  KRAS activation (30‐  vascular or pleural invasion. Trp53 inactivation 50%) . Esp in carcinogen induced tumors – Lepidic pattern ~ aerogenous  TRP53 inactivation dissemination or lepidic spread 149 150

Mouse Mammary Tumors Mouse Mammary Tumors Spontaneous Terminology C3H > other strains ‘Preneoplastic’ lesions – CBA developed as resistant control strain . HAN Hyperplastic alveolar nodules . Plaques BALB/c, 129 etc . MIN mammary intraepithelial nodules –GEM FVB/N get hyperplasia, EMT Adenoma How MMTV Bittner agent was discovered Carcinoma – Transmitted in milk – cytoplasmic inclusions – Reduced incidence by fostering Pattern descriptors – Endogenous Mtv’s involved in later tumors too 151 152Etiology descriptors

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Mouse Mammary Tumors MOUSE Mammary Tumors Patterns  Tumors  Hyperplasia Thelma Dunn 1958  C3H, etc  Pregnant? . Type A tumor   FVB/N –pituitary tt? MMHCC e.g. . = Adenocarcinoma, acinar, low grade, MMTV 

MMTV ‘spontaneous’ + MMTV Wnt Fgf3 activated GEM

153 154 http://tgmouse.compmed.ucdavis.edu/preprint/Kenney/Dunn1958.pdf

Mouse Mammary Tumors FVB/N Patterns Mammary hyperplasia  ‘neoplasia?  Dunn 1958 . Type B (Both /mixed)  MMHCC: . acinar, glandular, papillary, cystic, solid  MMTV ‘spontaneous’ +  Wnt Fgf3 activated GEM

155 http://tgmouse.compmed.ucdavis.edu/preprint/Kenney/Dunn1958.pdf 156

Mouse Mammary Tumors Mouse Mammary Tumors Patterns V Human Breast Tumors Dunn 1958 SIMILARITIES DIFFERENCES  . Carcinosarcoma  Molecular lesions Mouse MT met to lung. – Human met to regional L nodes  Some morphologies MMHCC:  Hu  more ductal Ca  . Carcinosarcoma Consistent with multi‐  Mouse MT have less fibrosis and hit kinetics Now: EMT inflammation  Metastatic in both  Most mouse MT are hormone Independent Carcinogen associated – ~1/2 of human breast Ca are . E.g. hydrocarbons hormone‐dependent. FVB/N + pituitaryT?  MMTV’ & Mtv’s in Mice – H hepaticus increased MT 157 http://tgmouse.compmed.ucdavis.edu/preprint/Kenney/Dunn1958.pdf Cf Thompson158 and Cardiff in a mouse model

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Mouse Liver Tumors WYD spontaneous Gross – nodules: single to multifocal coalescing Histology – Foci of cytoplasmic alteration . Eosinophilic + Clear cell > Basophilic or mixed – Adenomas more common than CA – Carcinomas may arise in adenomas – Hepatoblastomas may arise in Ca 159

Mouse Liver Tumors Liver tumors C3H, CBA > other strains; M>>F

http://dir.niehs.nih.gov/dirlep/liver2/MLIVER/mmenu.htm Maronpot 161

Mouse Liver Tumors Mouse Liver Tumors  Foci of cellular alteration – Eosinophilic, basophilic, clear, mixed  Hepatocellular Adenoma (Type A nodules /tumors) C3H, CBA, B6C3F1 – Well demarcated, usually < 10mm   Hepatocellular Carcinoma (Type B nodules/tumors) M > F – Trabecular, adenoid > solid patterns C3H up to 90% incidence in M @14m – Well to poorly differentiated – Usually poorly demarcated, > 10mm Hcs (Hepatocarcinogen sensitivity) loci  Hepatoblastoma usually arise in/near carcinomas Role of Helicobacter etc. Microbes? – GEM .  • http://dir.niehs.nih.gov/dirlep/liver2/MLIVER/mmenu.htm Maronpot A/J Liver tumors • Thoolen, Maronpot, et al. (2010). "Proliferative and Nonproliferative Lesions of the Rat and Mouse Hepatobiliary System. Tox Path. 2010;38:5S 163INHAND Nomenclature 164

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Profiling Profiling …

Not always a bad thing 1. Thymic Lymphoma b) AKR, NODscid, C58 Spontaneous tumors to expect : 2. B cell Lymphoma g) SJL/J (etc) SCC TCC MH 3. Plasma cell tumors (myeloma) (Harderian, myoepithelioma, c) BALB/c Rhabdomyoma etc ) Bernese Mtn dog Briard 4. Lung tumors a) A strains Standard poodle Scottie Flat coated Retreiver 5. Mammary tumors d) C3H strains Gordon Setter Westie Scottie Giant Schnauzer 6. Mammary Hyperplasia also Westie F) FVB/N EMT with pituitary tumors 7. Liver tumors d) C3H, CBA

http://research.nhgri.nih.gov/dog_genome/165 8. Cancer Resistant e) C57Bl/6 (DBA, CBA) 166

Mouse Pituitary Tumors … 5. 5 Mouse Tumors Most common /likely  Adenomas of pars distalis are more common than of pars intermedia.  Lymphoma; Lung; Liver; Mammary; Pituitary etc.  Aged females.  B6 is pretty resistant – is this good for our cancer studies?  Like rat may secrete prolactin CONCLUSIONS  well‐delineated  EXPECT THE EXPECTED – consider the unexpected  Compressive  PATHOLOGY for diagnosis  angiectatic or – Spontaneous expected vs important or unexpected cyst‐like spaces are common.  Don’t let expected conditions mess up your research – Don’t miss unexpected important phenotypes 167 168

More tumors MICE ‐ NEOPLASIA A/J: rhabdomyosarcoma  Diagnosis/es? Chimeras – Rhabdomyosarcoma . Extragonadal teratomas – Pleomorphic type Various Sporadic Gem  Likely Strains? . Leukemias, myeloid, erythroid . Hemangiosarcoma, etc sarcoma – A/J 70‐80% at >20mo . Endocrine – PITUITARY >> Adrenal, thyroid ‐ Pten etc – BALB/cByJ less freq . Salivary tumors, myoepithelioma, Harderian tumors,  Implicated mammary tumors, myeloma –BALB/c genes/mutations? – Dysf Im

Sher et al. 2011 Rhabdomyosarcomas in Aging A/J Mice. PLoS ONE 6(8): e23498. doi:10.1371/journal.pone.0023498 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0023498 169

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Chimeras (N0, F0) Extragonadal teratoma, teratocarcinoma

Chimera

171 172

Teratoma in B6, 129 chimera 8wo GEM

Swollen, fractured? tail  Sick but ‘NOT the Leukemia Genotype …. ‘?  Leukemic

173

Other Tumors … RAT Diagnosis/es? Diagnosis/es? – Thyroid follicular hyperplasia – Thyroid follicular hyperplasia – Adenoma? – Adenoma? Cause/s? – C cell hyperplasia – Pten mutation – Adenoma

In rats –also C Cell tumors Mice are NOT little http://www.goreni.org/ 175rats 176

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1 yo GEM 2yo GEM

Diagnosis/es? Diagnosis/es? – Thyroid follicular – Thyroid follicular hyperplasia hyperplasia – Adenoma – Adenoma Cause/s? Cause/s? – Pten mutation – Pten mutation  Look for ectopic thymus etc near thyroid

Other Tumors … WYD?

Diagnosis/es . Adrenal cortical hyperplasia . Subcapsular cell hyperplasia . adenoma

 In rats (& p53 deficient mice) Medullary tumors are more common – NOT little rats

1mm

179

Other Tumors – WYD? Hemangiosarcoma  Peliosis  Telangiectasis Sporadic  Hemangiosarcoma  Other? Trp53 mutations

185

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Mouse Sarcoma IHC Other Tumors –Skin – subcutis

Sarcoma, fibrosarcoma

FVB/N > others?

Ear tags etc.  Rehg & Ward (2012). Vet Path. For AB etc info: Trp53 mutations  http://journals.sagepub.com/doi/full/10.1177/0300985811429813 – NIEHS /NTP protocols:  https://www.niehs.nih.gov/research/resources/protocols/protocols‐ immuno/index.cfm 186

WYD? Mouse Clinical sign (s)?  Likely strain (s)? Diagnosis/es – Mammary tumor vs salivary tumor – Myoepithelioma ?  Likely strain? – BALB/c

 IHC For diagnosis?

188 Askjpc.org

Myoepithelioma Eureka –It Worked Mammary? Salivary? Likely strain(s)?  Rx  Control Less likely cause(s)?

 Bladder Cancer Xenograft  Selective killing of cancer cells by a small molecule targeting the stress response to ROS  Raj & al 2015 190 191

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Welfare breach prompts Nature to update policy on publishing Thanks! DANKE! animal experiments  Mice & GEM September 2015  Nadine Forbes That’s all folks!  Wendy Elza “As a result of this case, we are increasing the  MCP & core faculty amount of information we request from  LAM & Vet path trainees on authors. In experiments in which tumours are – NIH T32 RR0077022 & grown, we now require authors to include the maximal tumour size permitted by the  institutional animal‐use committee, and to Email [email protected]  Google Hopkins + Phenotyping state that this was not exceeded,” says the

193 Editorial. 196

Perdido Beach Mouse Perdido Beach Mouse Peromyscus polionotus trissyllepsis  Peromyscus polionotus Taylor & al 2015 trissyllepsis along entire vertebral column  Whats your diagnosis? – cervical, 5 of 38 (13%); thoracic, 16 (42%); lumbar, 13 (34%); and sacral, 10 (26%) – 10 (26%) mice had multiple primary masses. – Lung metastases in 13 of 38 (34%) mice. – 424 to 2170 days old, – Similar prevalence in males (n = 23 of 50; K. R. Taylor et al. Vet Pathol 2015;0300985815575051 198 Copyright © by American College of Veterinary Pathologists 46%) and females (n = 15 of 38; 39%).

Perdido Key beach mouse, DBA/2NNia C57Bl/6NNia chordoma.

3. Vertebra 4. Lung 5. Lung ORO 6. Lung PAS DBA/2NNia C57Bl/6NNia 7. Lung IHC – brachyury 8. Lung IHC –Ck AE1/AE3

K. R. Taylor et al. Vet Pathol 2015;0300985815575051 Copyright © by American College of Veterinary Pathologists Copyright © by American College of Veterinary Pathologists Harbison et al. Vet Pathol 2015;53:468-476

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DBA/2NNia B6D2F1 C57Bl/6NNia, DBA/2NNia B6D2F1

 Genotype Dependency  Effects of 40% CR.a B6D2F1 DBA/2NNia  Common Lesions in Male and Female  1989-90

Copyright © by American College of Veterinary Pathologists Copyright © by American College of Veterinary Pathologists C. E. Harbison et al. Vet Pathol 2015;53:468-476 C. E. Harbison et al. Vet Pathol 2015;53:468-476 Figures 9-12

C57Bl/6NNia, Orbital Venous Sinus or DBA/2NNia Plexus? SINUS B6D2F1  Recommended approach for blood collection ? (AFTER Approval by SINUS IACUC/ethical committee)

A. Dorsal to the eye

B. Lateral canthus PLEXUS C. Medial canthus Timm. 1980. Synapse.

Cecum location and IP injections STRAIN influences on Cerebral Vasculature and Stroke Models (etc.

 microvascular injection with carbon black stained latex.  Differences in MCA distribution indicated by anastomosis Where to inject ? points between middle cerebral • Lower right artery (MCA) and quadrant…. anterior cerebral artery (ACA) Murat et al Copyright © 2016 Laboratory Animals Limited Maeda et al 1988 © Lippincott‐Raven Publishers. Published by Lippincott Williams & Wilkins, Inc. http://journals.sagepub.com/doi/full/10.1177/0023677216658916

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Experimental ‘Stroke’ susceptibility & STRAIN influences Cerebral Vasculature Cerebral vascular anatomy and Stroke Models (etc.)

 C57Bl/6 usually more susceptible to focal and global ischemia than CBA, DBA/2, MF1, 129 Filament C57BL/6NCrj CBA/jNCrj DBA/2NCrj occlusion – substrains often not  C57Bl/6J (Ola) ICA ACA specified  – 1/10 mice had a complete circle of PcomA – CCO, Filament etc models Willis – 6/9 had one posterior communicating artery absent  + variation among C57Bl/6 – 3/9 were deficient for both PcomAs. – McColl et al 2004 VA Gerbil Rat Kitagawa et al 1998. http://journals.sagepub.com/doi/pdf/10.1097/00004647‐199805000‐ 00012

STRAIN influences Cerebral Vasculature and Stroke Models (etc.) C57BL/6J Portal systemic shunts CD31 for endothelium D2–40 for lymphatics  Bilateral CCA  Diagnosis/es? occlusion x15 min+ – Portal vein hypoplasia reperfusion x7d. – Portal‐systemic shunt – MAP2 immunoreactivity  Clinical/gross findings ? (microtubule‐associated – None protein 2) level of  caudoputamen and Research relevance hippocampus, – abnormal neurochemical profile in ~25% – Cresyl violet staining in –  cerebral glutamine & right hippocampus CA 1 myo‐inositol  – Patent PcomA completes Other strains? C57BL/6NCrj DBA/2NCrj circle of Willis, and – 129/Ola – schistosome maintainsKitagawa et al perfusion… 1998. http://journals.sagepub.com/doi/pdf/10.1097/00004647‐199805000‐ resistant… 00012

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