Pathology of the Posterior Segment in Ocular Toxicology

Pathology of the Posterior

Segment in Ocular

Toxicology Studies

Steven D. Sorden, DVM, PhD, Diplomate ACVP

Covance Laboratories Inc., Madison, WI

Please email questions to

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Outline

Anatomy

Topographic & species variations

Antemortem Evaluation of the Posterior Segment

Indirect Ophthalmoscopy

Fluorescein Angiography

Optical Coherence Tomography (OCT)

Electroretinography (ERG)

Postmortem Evaluation of the Posterior Segment

Histology/Methods

Microscopic Findings


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Anterior

Segment

Posterior

Segment

Optic

Nerve

Optic

Disc

Retina

Choroid

Sclera

Abbreviations - Retina ILM Inner limiting membrane

NFL Nerve fiber layer

GCL Ganglion cell layer

IPL Inner plexiform layer

INL Inner nuclear layer

OPL Outer plexiform layer

ONL Outer nuclear layer

OLM Outer limiting membrane

PRL Photoreceptor layer (rods & cones)

IS Inner segment of photoreceptor layer

OS Outer segment of photoreceptor layer

RPE Retinal pigment epithelium Please email questions to

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Vitreous

GCL

RPE

INL

ONL

PRL

Basic Retinal Anatomy

http://webvision.instead-technologies.com

NFL

Vitreous

NFL/GCL

IPL

INL

ONL

PRL

Choroid

Sclera

OPL

Extraocular

muscle

Basic Retinal Anatomy (rat)

RPE

IS

IPL

INL

ONL

Choroid

Sclera

OPL

OS

Basic Retinal Anatomy (rat)

Specialized Topographic Regions

of the Retina

Increased cone &/or ganglion cell density

Increased visual acuity

Visual Streak – dog, rabbit, pig

Nasotemporal zone of increased ganglion cell density

Area Centralis – dog, cat, pig

Focal zone of maximal cone & ganglion cell density

Superior temporal, center of visual field

Dog A.C. recently shown to contain “foveal bouquet”

Macula lutea with fovea centralis – NHP, humans

A type of area centralis


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Rabbit

Visual Streak

Peripheral

Retina

GCL

GCL

Tapetum

GCL

Canine Retina,

Area Centralis Canine Retina

GCL


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Foveal bouquet

Tapetum

INL

ONL

GCL

Canine Area Centralis


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RPE

Tapetum lucidum

Canine Tapetal & Nontapetal Choroid

Cynomolgus Monkey

Posterior Segment –

Horizontal Section

Nasal

Temporal

Macula

Fovea

Vitreous

**GCL

IPL

INL

ONL

PRL

Choroid

OPL

NHP Retinal Anatomy - Macula

NFL

RPE

IS

INL

ONL

Choroid

OPL

OS

NHP Retinal Anatomy

Rod Pedicles &

Cone Spherules

OLM

Fovea


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Rabbit Eye – Mid-Sagittal

Superior

Inferior

*

Rabbit Optic Nerve/Retina

Vitreous Physiologic

cup in optic disc

Optic Nerve

Rabbit – Medullary Ray

Rabbit – Merangiotic Retinal Vasculature

Antemortem Evaluation –

Indirect Ophthalmoscopy

Conducted after

dilation of pupil

with short-acting

mydriatic

(tropicamide)

Evaluates: Vitreous

Fundus

Retina

Vessels

Optic disc

Choroid

Courtesy of Robert J. Munger, DVM, DACVO


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Normal Rabbit Fundus New Zealand Red Rabbit New Zealand White Rabbit

Courtesy of Robert J. Munger, DVM, DACVO Please email questions to

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Normal Primate Fundus


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Correlation of Ophthalmoscopy &

Histopathology

Ophthalmoscopy provides images of vitreal aspect

of most of the retina at multiple time points, vs. 4-5

micron sections at a single time point

Images/drawings of ophthalmoscopic findings are

critical to selection of regions for histopathology

Ophthalmoscopy

Requires transparent anterior segment & vitreous

Does not image peripheral retina

Is less sensitive, ie., cannot detect subtle findings that

can be detected microscopically


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Fluorescein Angiography (FA)

Evaluation of the retinal and choroidal circulation

via intravenous injection of sodium fluorescein

In toxicology, primarily used to detect

hyperfluorescence due to neovascularization/

capillary leakage

Procedure

Sedate, administer IV fluorescein, and

obtained timed sequential photographs


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FA – Hyperfluorescence – Laser-Induced Choroidal Neovascularization


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Laser-Induced Chorioretinal Scar

Chorioretinal Scar/Adhesion

Detached retina RPE

Laser-Induced Chorioretinal Scar

Optical Coherence Tomography

(OCT)

Analogous to ultrasound, but measures

time delay of light instead of sound

Based on interferometry

Spectral domain most common (sdOCT)

Cross-sectional or 3D images

Resolution approaches light microscopy

Axial resolution: 4-5 um


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Value of OCT for nonclinical studies

Baseline screening for background lesions

Repeated evaluation allows: Evaluation of progression or

regression May alleviate need for

interim sacrifices. Reduces animals, time and $

Complements other endpoints, especially if fundus view is degraded

Sensitive biomarker applicable to clinical setting - translatable

CZMI

OSODHigh Definition Images: HD 5 Line Raster

Signal Strength:

Technician:

Exam Time:

Exam Date:

Doctor:

Gender:

DOB:

ID:

Name:

10/10

Operator, Cirrus

8:37 AM

6/25/2012

Female

2/3/2000

CZMI401459494

8255607, I01135 - IM113878

Length:Spacing:Scan Angle: 3 mm0 mm0°

Doctor's Signature OSODSW Ver: 5.2.1.12Copyright 2011Carl Zeiss Meditec, IncAll Rights Reserved

Page 1 of 1

Comments


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Multiple Imaging Modes - Retinal nerve fiber layer tomogram in normal and glaucomatous NHP eyes

Normal

Glaucomatous

Note loss of RNFL

RNFL


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Correlation of OCT & Histopathology

Correlation is often good, but like ophthalmoscopy,

correlation requires

Sampling of appropriate region for

histopathology

Correlation of OCT findings from exam

temporally closest to sacrifice

Mild OCT findings may lack microscopic correlate

TEM or other methods may be required

Mild microscopic findings may lack an OCT

correlate


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Electroretinography (ERG)

In-situ index of retinal electrophysiology

Direct translational application to humans

a-wave = photoreceptor response

to light stimulus

b-wave = responses of bipolar

cells (mostly) + Müller cells

Evaluate under scotopic (dark-adapted [rods]) and photopic (light-adapted [cones]) conditions

Ganglion cell response is NOT

measured

ERG response ≠ visual perception


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Electroretinography

Full-field “flash” ERG (FERG) – most common

Sensitive to factors that globally affect

photoreceptors and bipolar cells

Multifocal Electroretinography (mERG)

Detect localized loss of function

Pattern reversal ERG (PERG)

Assesses ganglion cell function

Visual Evoked Potential (VEP)

pathway from ganglion cells to visual cortex


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Correlation of ERG & Histopathology

Correlation may be poor, because FERG is

insensitive to localized loss of function

FERG may be normal in animals with

marked microscopic lesions, especially if

focal/multifocal.

FERG is not affected by lesions of inner

retina and optic nerve.

mERG, PERG, VEP may correlate.


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Postmortem Evaluation of the

Retina

Histology/Methods

Immunohistochemistry

Electron Microscopy

Microscopic Findings

Spontaneous/Background Findings

Procedure-Related Findings

Test Article-Related Findings

Immune-mediated


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Postmortem Evaluation of the Retina

Collection - ASAP following euthanasia

Ink at 12 o’clock, injection sites, etc.

Fixation

Immersion

Davidson’s/Modified Davidson’s

corneal artifacts, especially in rabbits

10% neutral buffered formalin, +/- glutaraldehyde –

better corneal & optic nerve morphology

Transmission electron microscopy (TEM) – fixatives

containing glutaraldehyde - Karnovsky’s, etc.

Injection – to replace collected vitreous

Upper body perfusion (TEM) ?? Please email questions to

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Optic Nerve – Longitudinal

Davidson’s Fixative –

Artifactual Vacuoles


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Optic Nerve – Transverse – 10%

Neutral Buffered Formalin


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Trimming

Standard vertical trim to include optic disc

May trim NHP horizontal to include fovea &

optic disc in same section. Or multiple vertical

sections.

Each half or smaller portion of globe = calotte.

Deep cassettes to minimize artifact.

Additional trims of injection sites, lesions,

inferior calotte to visualize test article in

vitreous. Please email questions to

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NHP Optic Disc – nonhorizontal trim

Macula not present.


Processing/Sectioning

Paraffin, 4-5 µm sections, hematoxylin &

eosin or immunohistochemistry

For NHP, sections should include fovea

Focal lesions may require serial

sections & multiple blocks

Experienced technicians are critical

Plastic, 0.5 – 1 µm sections for improved

morphology, esp. photoreceptors

Transmission electron microscopy (TEM) Please email questions to

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Plastic-embedded rat retina –

1 µm section, toluidine blue stain

Unlike routine H&E, stacks of

discs visible in outer segment


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Arrows indicate

cone nuclei in

ONL.

Smaller, darker

nuclei are rod

nuclei.

Plastic-embedded

NHP retina –

1 µm section,

toluidine blue stain


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Transmission electron micrograph – rod

outer segments - NHP


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Immunohistochemistry


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GFAP (glial fibrillary

acidic protein)

expression in NFL

Immunohistochemistry – RPE65

Immunohistochemistry Colabeling for S-cones & transfected green

fluorescent protein (RPE) in mouse retina

Spontaneous Retinal Findings

Recognition is critical

Must differentiate from test article-induced

findings

Some may be exacerbated by test articles

Eg., light-induced outer retinal degeneration if

test article causes mydriasis


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NHP - Mononuclear cell infiltrates in

ciliary body/choroid – common

background finding

NHP – Glial Nodule & Cystic Retinal

Degeneration at Ora Serrata


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Light-Induced Retinal Degeneration –

Albino Rodents

NORMAL

Loss of photoreceptors.

Fewer nuclei in outer

nuclear layer.

AFFECTED


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Bilateral Optic Atrophy in

Macaques Reported sporadically in rhesus and

cynomolgus macaques

Idiopathic “atrophy” adopted by authors, but problem may be developmental (onset and progression not documented)

FERGs normal, MERGs and PERGs affected to some degree

Temporal pallor of ONH, thinning of temporal RNFL (papillomacular bundle),

No behavioral issues


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Bilateral Optic Atrophy in Macaques

Fovea Fovea Ganglion

Cell Layer Atrophy of

Ganglion

Cell Layer

AFFECTED NORMAL

Optic Nerve

Atrophy of Temporal

Portion of Optic Nerve


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Atrophy of Temporal

Portion of Optic Nerve

Bilateral Optic Atrophy in Macaques

(In brain, corresponding reduction

of nuclei in lateral geniculate) Please email questions to

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Another (rare) Spontaneous Finding - Outer

Retinal Degeneration in a Mauritius Cynomolgus

Monkey

Inner Nuclear Layer

Outer Nuclear Layer

Outer Plexiform Layer

RPE RPE


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Procedure-Related Retinal Findings

Intravitreal injection

Focal fibrosis etc. in pars plana at injection site

Rarely hemorrhage in vitreous, retina

Laser sites – chorioretinal scars/adhesions

Subretinal injection – retinal detachment

Often transient

RPE hypertrophy/hyperplasia/hyperpigmentation

Loss/attenuation of photoreceptors

Fluid/cells in subretinal space


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NHP – Subretinal Injection Site (fovea)

NHP – Subretinal Injection Site (fovea)

RPE hypertrophy, hyperplasia, and

hyperpigmentation

NHP – Subretinal Injection Site

RPE hypertrophy & hyperpigmentation

Rabbit – Loss of Photoreceptors at Subretinal

Injection Site

Hypertrophy of RPE and photoreceptor loss –

consistent with true retinal detachment Please email questions to

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Test Article-Related Retinal Findings

Retinal pigment epithelium Hypertrophy/accumulation of metabolites

(autofluorescent)

Photoreceptors Degeneration – reversible loss of IS/OS

Necrosis - irreversible

Ganglion cells Degeneration, loss, accumulation of metabolites

Immune-mediated Eg., Intravitreal biologics – often humanized

Perivascular infiltrates of lymphocytes, plasma cells, macrophages – correlate with perivascular sheathing observed via ophthalmoscopy


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Immune-Mediated Retinal Findings Most commonly observed following repeated

intravitreal injection of humanized molecules in NHP and rabbits

Indirect Ophthalmoscopy Vitreous Cells

Perivascular Sheathing

Variable incidence, but often low in NHP Lack of a dose-response

Incidence increases with frequency of dosing

+/- correlation with anti-drug antibodies in affected individuals

Manifestations of immunogenicity in preclinical studies are generally not predictive of immunogenicity/hypersensitivity in humans


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NHP – Immune-Mediated Inflammation -

Intravitreal Humanized Monoclonal Ab –

Perivascular Cuffs of Leukocytes


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Artifact


Intravitreal Humanized Monoclonal Ab –

Vitreous Cells


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Artifact


Intravitreal Humanized MAb – Perivascular

Cuffs of Leukocytes – Optic Disc Please email questions to

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Fovea - unremarkable


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Iris distortion due to

collection of vitreous at necropsy

Increased amorphous eosinophilic material

In vitreous – test material

Granulomatous Response to Intravitreal

Injection of Insoluble Vehicle

Bonus Topic – Paraocular Gland

Lesions Induced by Blood

Collection in Rabbits

Medial ear artery catheters may be used

when multiple blood collections are required

in 8-24 hour window (toxicokinetic samples)

instead of multiple jugular venipunctures.


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Multifocal necrosis of rabbit lacrimal gland


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Necrosis of rabbit lacrimal gland


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Necrosis of rabbit Harderian gland


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Paraocular Gland Necrosis –

Proposed Pathogenesis

Although thrombi not identified in sections, lesions are morphologically consistent with infarcts and only seen in catheterized rabbits.

Flushing of catheters with saline prior to blood collections likely propels microthrombi formed between blood collections retrograde into the transverse facial or maxillary arteries via the auricular arteries.

Thromboemboli likely lodge in arterial branches that supply Harderian and lacrimal glands (and mandibular salivary glands) infarction.


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References Beltran WA, Cideciyan AV, Guziewicz, et al. Canine retina has a primate fovea-

like bouquet of cone photoreceptors which is affected by inherited macular degenerations. PLoS One. 2014 Mar 5;9(3):e90390

De Vera Mudry MC, Kronenberg S, et al. Blinded by the Light: Retinal Phototoxicity in the Context of Safety Studies. Toxicol Pathol 41:813-825, 2013.

Dubielzig RR, Leedle R, Nork TM, VerHoeve JA, Christian BJ. Bilateral Optic Atrophy: A Background Finding in Cynomolgus Monkeys Used in Toxicologic Research. Invest Ophthalmol Vis Sci 2009;50:E-Abstract 5344.

Fortune B, Wang L, Bui BV, Burgoyne CF, Cioffi GA. Idiopathic Bilateral Optic Atrophy in the Rhesus Macaque. Invest Ophthalmol Vis Sci 2005;46:3943-56.

Lee S-F, Sorden SD, Dunn DG, Sonnentag PJ, Dwyer AJ. Ocular Effects of Blood Collection Techniques in Rabbits. Abstract #3039, Invest Ophthalmol Vis Sci 2013.

Mecklenburg L, Schraermeyer U. An overview on the toxic morphological changes in the retinal pigment epithelium after systemic compound administration. Toxicol Pathol 35:252-267, 2007.

Render JA, Schafer KA, Altschuler RA. Special Senses: Eye and Ear. In: Toxicologic Pathology: Nonclinical Safety Assessment, Sahota PR, Popp JA, Hardisty JF, Gopinpath C, eds, CRC Press, Boca Raton, FL, 2013, p. 945.

Sato J, Doi T, Kanno T, Wako Y, Tsuchitani M, Narama I. Histopathology of Incidental Findings in Cynomolgus Monkeys (Macaca fascicularis) Used in Toxicity Studies. J Toxicol Pathol 2012;25:63-101.

Weir and Collins (eds). Assessing Ocular Toxicology in Laboratory Animals. Humana Press, New York, NY, 2013.

Documents

Pathology of the Posterior Segment in Ocular Toxicology