The mouse eye: ocular phentyping
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The Mouse Eye: Ocular Phenotyping (in 15 minutes or less) Gillian C. Shaw, DVM, MS September 28, 2013 [email protected]
Transcript of The mouse eye: ocular phentyping
The Mouse Eye: Ocular Phenotyping (in 15 minutes or less)
Gillian C. Shaw, DVM, MS September 28, 2013
Disclaimer
• Probably people in the room are more qualified than me to give this lecture!
• Please speak up and correct me if I say something that is inaccurate
• Or add Rdbits where you feel they’re needed
Mouse models of eye disease
• Can be used to model many (but not all) eye diseases that are relevant to humans
• They lack a macula & fovea, so exact macular diseases can’t be modeled – Can induce lesions in the mouse reRnas that are relevant to various macular diseases in humans (ex. choroidal neovascularizaRon)
• Large lens & small globe size make treatment tesRng difficult (injecRons, implants)
Mouse models of eye disease
• Spontaneous & induced geneRc models – Rd1 mouse, knock-‐out/knock-‐in/transgenic
• Induced/environmental disease models
– O]en combined with geneRc models to discern pathogenesis of a disease
EvaluaRon Methods
• Antemortem – Morphological:
• Gross exam • Slit lamp exam • Fundus exam
– Indirect ophthalmoscopy – OpRcal Coherence
Tomography
• MRI/CT scan
– FuncRonal • OptokineRc device • ElectroreRnogram, VEPs • Fluorescein angiography
• Postmortem/Morphology – H&E – EM – IHC – ReRnal flat-‐mount
• Cell specific markers • Vascular lesions
– Dissociated whole reRnal cultures or immuno-‐selected cells
ReRnal Analysis
• ReRnal Imaging Microscopy System allows ‘in-‐vivo microscopy’ • White light imaging mice and rats, fluorescein angiography,
diabeRc reRnopathy, reRnoblastoma, reRniRs pigmentosa, choroidal neovascularizaRon & anterior segment slit-‐lamp
• Live animal GFP & YFP fluorescent studies also possible
R-4300 for Greatest Depth, R-2200 for Greatest Resolution
R4300 Whole Eye R2200 Posterior R2200 Anterior R2200 Periphery
Histology
SD-‐OCT
7
Mouse
MagneRc Resonance Imaging (MRI)
Bogom images: Analysis of postnatal eye development in the mouse with high-‐resoluRon small animal magneRc resonance imaging. Tkatchenko TV, Shen Y, Tkatchenko AV. Invest Ophthalmol Vis Sci. 2010 Jan;51(1):21-‐7.
ReRnal FuncRon: ElectroreRnogram
(ERG)
ON ON
3rd order neurons
Cone Rod
Bipolar cells OFF
Visual funcRon: OptokineRc Device
Cell transplantaRon as a treatment for reRnal disease. Lund RD, Kwan AS, Keegan DJ, Sauvé Y, Coffey PJ, Lawrence JM. Prog ReRn Eye Res. 2001 Jul;20(4):415-‐49.
Normal Mouse Eye
hgp://www.deltagen.com/target/histologyatlas/HistologyAtlas.html
Whole Globe: GeneRc Models
• Buphthalmos (whole eye)
• Axial myopia (single axis) • Anophthalmos
• Microphthalmos Increased ocular levels of IGF-‐1 in transgenic mice lead to diabetes-‐like eye disease. Jesús Ruberte, Eduard Ayuso, Marc Navarro, Ana Carretero, Víctor Nacher, Virginia Haurigot, Mónica George, CrisRna Llombart, Alba Casellas, CrisRna Costa, Assumpció Bosch, FaRma Bosch. J Clin Invest. 2004; 113(8):1149–1157.
Cataracts and microphthalmia caused by a Gja8 mutaRon in extracellular loop 2. Xia CH, Chang B, Derosa AM, Cheng C, White TW, Gong X. PLoS One. 2012;7(12):e52894.
IGF-‐1 ocular overexpression
Gja8 (connexin) mutant
Anophthalmia & Microphthalmia • C57BL mice known for small or absent eyes • Frequency varies from 1 to 10% depending on background strain – Females and right eyes affected more o]en than males & le] eyes
• Gene(s) have not been idenRfied • Also more suscepRble to ocular infecRons because of abnormal flushing of ocular surface
• Microphthalmia and associated abnormaliRes in inbred black mice. Smith RS, Roderick TH, Sundberg JP. Lab
Anim Sci. 1994 Dec;44(6):551-‐60.
hgps://database.riken.jp/sw/en/microphthalmia_model_mouse/crib16s27rib16s2i/
Induced Corneal Injury • Alkali burn
– Bugon of filter paper soaked with 1N NaOH applied to cornea of anestheRzed mice
hgp://www.deltagen.com/target/histologyatla
s/HistologyAtlas.html
Effects of topical administraRon of 12-‐methyl tetradecanoic acid (12-‐MTA) on the development of corneal angiogenesis. Cole N, Hume EB, Jalbert I, Vijay AK, Krishnan R, Willcox MD. Angiogenesis. 2007;10:47–54.
Normal Cornea
Induced Corneal Injury • Fusarium keraRRs model
– Contact lens with adherent Fusarium applied to abraded mouse eye
hgp://www.deltagen.com/target/histologyatla
s/HistologyAtlas.html
A murine model of contact lens-‐associated Fusarium keraRRs. Sun Y, Chandra J, Mukherjee P, Szczotka-‐Flynn L, Ghannoum MA, et al. Invest Ophthalmol Vis Sci. 2009;51:1511–1516.
Normal Cornea
Modeling Dry Eye (keratoconjuncRviRs sicca)
• Experimental reduced lacrimaRon – Scopolamine patch applied to tail
– Botulinum toxin injecRon into lacrimal gland
– with or without environmental change (low humidity, increased air movement) Inflammatory cytokine expression on the ocular surface in the Botulium toxin
B induced murine dry eye model. Zhu L, Shen J, Zhang C, Park CY, Kohanim S, Yew M, Parker JS, Chuck RS. Mol Vis. 2009;15:250-‐8.
Fluorescein staining (punctate green) on damaged corneal surface (A, B, C) compared to normal (D, E, F) a]er botulinum toxin induced dry eye
Fuchs Endothelial Corneal Dystrophy Model
• Mice with knock-‐in mutaRon of the alpha 2 collagen 8 gene – Progressive alteraRons in
corneal endothelial morphology
– Cell loss & basement membrane gugae
An alpha 2 collagen VIII transgenic knock-‐in mouse model of Fuchs endothelial corneal dystrophy shows early endothelial cell unfolded protein response and apoptosis. Jun AS, Meng H, Ramanan N, Maghaei M, ChakravarR S, Bonshek R, et al. Hum Mol Genet. 2012;21:384–393.
WT WT Mut Mut Corneal orientaRon
UnintenRonally induced cataracts
Time course of cold cataract development in anestheRzed mice. Bermudez MA, Vicente AF, Romero MC, Arcos MD, Abalo JM, Gonzalez F. Curr Eye Res. 2011 Mar;36(3):278-‐84.
Acute reversible cataract induced by xylazine and by ketamine-‐xylazine anesthesia in rats and mice. Calderone L, Grimes P, Shalev M. Exp Eye Res. 1986 Apr;42(4):331-‐7.
CAN be unilateral. ARE reversible.
• N-‐acetyl-‐p-‐benzoquinone imine (NAPQI) = acetaminophen metabolite – Intracameral (AC) injecRon of
NAPQI elicits increase in free Ca2+ in lens epithelium, calpain acRvaRon & lens opacificaRon
• UVR-‐B induced cataract development
Naphthoquinone-‐Induced cataract in mice: possible involvement of Ca2+ release and calpain acRvaRon. Qian W, Shichi H. J Ocul Pharmacol Ther. 2001 Aug;17(4):383-‐92.
UVR-‐B induced cataract development in C57 mice. Meyer LM, Söderberg P, Dong X, Wegener A. Exp Eye Res. 2005 Oct;81(4):389-‐94.
The lens is prone to arRfact in processing for histologic imaging, so imaging the lens antemortem with a slit lamp and/or imaging dissected lenses immediately a]er euthanasia is most reliable.
IntenRonally induced cataracts
WT
GeneRc and allelic heterogeneity of Cryg mutaRons in eight disRnct forms of dominant cataract in the mouse. Graw J, Neuhäuser-‐Klaus A, Klopp N, Selby PB, Löster J, Favor J. Invest Ophthalmol Vis Sci. 2004 Apr;45(4):1202-‐13.
Cataract Models WT Mut
Glaucoma
• What is glaucoma? – More complicated than just elevated intraocular pressure
– Loss of reRnal ganglion cells (RGCs) with opRc nerve degeneraRon/atrophy
• Induced Models – Bead injecRon, dexamethasone injecRon
• GeneRc Models – DBA/2J mouse, MYOC mutant mouse
IOP Measurement in Mice
• AC needle vs tonolab • AnestheRcs affect IOP (inhalaRonal) • Hold mouse too Rght affects IOP
• Corneal issues affect Tonolab CalibraRon of the TonoLab tonometer in mice with spontaneous or experimental glaucoma. Pease ME, Cone FE, Gelman S, Son JL, Quigley HA. Invest Ophthalmol Vis Sci. 2011 Feb 22;52(2):858-‐64.
Induced glaucoma: bead injecRon
DifferenRal suscepRbility to experimental glaucoma among 3 mouse strains using bead and viscoelasRc injecRon. F.E. Cone, S.E. Gelman, J.L. Son, M.E. Pease, H.A. Quigley. Exp. Eye Res., 91 (2010), pp. 415–424.
Bead injected, axon loss in opRc nerve
Non-‐injected eye, normal axon density in opRc nerve
CalibraRon of the TonoLab tonometer in mice with spontaneous or experimental glaucoma. Pease ME, Cone FE, Gelman S, Son JL, Quigley HA. Invest Ophthalmol Vis Sci. 2011 Feb 22;52(2):858-‐64.
ReRnal Disease
• Neural reRna (reRnal degeneraRon, RD) – Direct effect on
reRnal neurons – ReRnal pigmented
epithelium (RPE) with secondary effects on neural reRna
• Vascular diseases – DiabeRc reRnopathy
(DR) – Age-‐related macular
degeneraRon (AMD)
hgp://www.bio.miami.edu/tom/courses/protected/bil265/reRna.jpg
(Neural) ReRnal DegeneraRons
• Induced – Light induced reRnal degeneraRon – N-‐methyl-‐N-‐nitrosurea (MNU)
• GeneRc Models – Spontaneous – Transgenic – Many examples of both
Induced ReRnal DegeneraRons • Light induced reRnal degeneraRon
– Bright light exposure with dilated pupils
The Rpe65 Leu450Met variaRon increases reRnal resistance against light-‐induced degeneraRon by slowing rhodopsin regeneraRon. Wenzel A, Reme CE, Williams TP, Hafezi F, Grimm C. J Neurosci. 2001;21:53–58.
Balb/C No exposure
Balb/C 8 hrs light exposure
Induced ReRnal DegeneraRons • N-‐methyl-‐N-‐nitrosurea (MNU)
– Single systemic dose causes photoreceptor apoptosis and reRnal degeneraRon within days
Animal models for reRniRs pigmentosa induced by MNU; disease progression, mechanisms and therapeuRc trials. Tsubura A, Yoshizawa K, Kuwata M, Uehara N. Histol Histopathol. 2010 Jul;25(7):933-‐44.
GeneRc reRnal degeneraRon: Rd1 (Pde6brd1) mutaRon
• Develop normal photoreceptors that then rapidly degenerate in the 3rd post-‐natal week – Complete blindness by 4 weeks of age
• C3H/HeJ, CBA/J, FVB/NJ, SJL/J, SWR/J – Many others – FVB/N of parRcular concern because they are o]en used to make targeted mutants because of their large pronucleus
• hgp://eyemutant.jax.org/index.html – Complete list of strains carrying the rd1 mutaRon
ReRnal DegeneraRon Models
ReRnal degeneraRon mutants in the mouse. Chang B, Hawes NL, Hurd RE, Davisson MT, Nusinowitz S, Heckenlively JR. Vision Res. 2002 Feb;42(4):517-‐25.
Vascular eye disease models
• Age-‐related macular degeneraRon – Dry = drusen deposiRon between choroid & RPE
– Wet = vascular problem caused by unhappy (hypoxic) cell signalling
• DiabeRc reRnopathy & ReRnopathy of prematurity – Pathogenesis similar to wet AMD
hgp://jirehdesign.com/eye-‐illustraRons/eye-‐condiRons-‐illustraRons/co0069.html
Age-‐related “Macular” degeneraRon (AMD) models
• Mice make imperfect animal models of AMD because they lack a macula – i.e. no perfect single model available
• But many hallmarks of AMD in humans can be modeled in various mouse models
• There are many examples of geneRc and induced (and combinaRons of the two) models
• Example: – SOD1 -‐/-‐ mouse (superoxide dismutase) [wet AMD]
SOD1 deficient mice
Drusen, choroidal neovascularizaRon, and reRnal pigment epithelium dysfuncRon in SOD1-‐deficient mice: a model of age-‐related macular degeneraRon. Imamura Y, Noda S, Hashizume K, Shinoda K, Yamaguchi M, Uchiyama S, Shimizu T, Mizushima Y, Shirasawa T, Tsubota K. Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11282-‐7.
Choroidal neovascularizaRon (CNV) = vessels invade through Bruch’s membrane and into the RPE & reRna. CNV vessels are abnormal and leaky.
DiabeRc ReRnopathy
• C57BL/6JIns2Akita mice (mutn in insulin 2 gene) • diffusion of fluorescently labeled BSA into the adjacent
parenchyma of a blood vessel • (le] image = cross secRon, 2 right images = reRnal flat mounts)
Pigment epithelium-‐derived factor (PEDF) pepRde eye drops reduce inflammaRon, cell death and vascular leakage in diabeRc reRnopathy in Ins2(Akita) mice. Liu Y, Leo LF, McGregor C, GriviRshvili A, Barnstable CJ, Tombran-‐Tink J. Mol Med. 2012 Dec 20;18:1387-‐401.
Untreated Akita mouse Untreated Akita mouse Treated Akita mouse
Oxygen-‐induced ReRnopathy (OIR) Netrin-‐1 overexpression in oxygen-‐induced reRnopathy correlates with breakdown of the blood-‐reRna barrier and reRnal neovascularizaRon. Tian XF, Xia XB, Xiong SQ, Jiang J, Liu D, Liu JL. Ophthalmologica. 2011;226(2):37-‐44.
The mouse reRna as an angiogenesis model. Stahl A, Connor KM, Sapieha P, Chen J, Dennison RJ, Krah NM, Seaward MR, Willeg KL, Aderman CM, Guerin KI, Hua J, Löfqvist C, Hellström A, Smith LE. Invest Ophthalmol Vis Sci. 2010 Jun;51(6):2813-‐26.
P7 mice are exposed to 75% oxygen, which induces loss of immature reRnal vessels and slows development of the normal reRnal vasculature, leading to a central zone of vaso-‐obliteraRon (VO). A]er returning mice to room air at P12, the central avascular reRna becomes hypoxic, triggering both normal vessel regrowth and a pathologic formaRon of extrareRnal neovascularizaRon (NV).
Strain Differences
• Inbred mice can carry background disease that can influence ocular phenotype: – 1. Systemic disease can affect the eye – 2. Known geneRc defects (such as Pde6brd1) – 3. Congenital abnormaliRes that may be polygenic (microphthalmia in black mice)
– 4. SuscepRbility genes that alter the response to an external sRmulus
Richard S. Smith (2001). SystemaGc EvaluaGon of the Mouse Eye: Anatomy, Pathology, and Biomethods (Research Methods For Mutant Mice). (Simon W. M. John, Patsy M. Nishina, John P. Sundberg, Eds). New York: CRC Press.
QuesRons?
