pathology of the posterior segment in ocular toxicology...
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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
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Satish.Panchal@sunpharma.com
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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Satish.Panchal@sunpharma.com
Rabbit
Visual Streak
Peripheral
Retina
GCL
GCL
Tapetum
GCL
Canine Retina,
Area Centralis Canine Retina
GCL
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Satish.Panchal@sunpharma.com
Foveal bouquet
Tapetum
INL
ONL
GCL
Canine Area Centralis
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Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
Normal Rabbit Fundus New Zealand Red Rabbit New Zealand White Rabbit
Courtesy of Robert J. Munger, DVM, DACVO Please email questions to
Satish.Panchal@sunpharma.com
Normal Primate Fundus
Courtesy of Robert J. Munger, DVM, DACVO Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Courtesy of Robert J. Munger, DVM, DACVO Please email questions to
Satish.Panchal@sunpharma.com
FA – Hyperfluorescence – Laser-Induced Choroidal Neovascularization
Courtesy of Robert J. Munger, DVM, DACVO Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
Multiple Imaging Modes - Retinal nerve fiber layer tomogram in normal and glaucomatous NHP eyes
Normal
Glaucomatous
Note loss of RNFL
RNFL
Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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.
Please email questions to
Satish.Panchal@sunpharma.com
Postmortem Evaluation of the
Retina
Histology/Methods
Immunohistochemistry
Electron Microscopy
Microscopic Findings
Spontaneous/Background Findings
Procedure-Related Findings
Test Article-Related Findings
Immune-mediated
Please email questions to
Satish.Panchal@sunpharma.com
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
Satish.Panchal@sunpharma.com
Optic Nerve – Longitudinal
Davidson’s Fixative –
Artifactual Vacuoles
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Satish.Panchal@sunpharma.com
Optic Nerve – Transverse – 10%
Neutral Buffered Formalin
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Satish.Panchal@sunpharma.com
Postmortem Evaluation of the Retina
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
Satish.Panchal@sunpharma.com
NHP Optic Disc – nonhorizontal trim
Macula not present.
Postmortem Evaluation of the Retina
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
Satish.Panchal@sunpharma.com
Plastic-embedded rat retina –
1 µm section, toluidine blue stain
Unlike routine H&E, stacks of
discs visible in outer segment
Please email questions to
Satish.Panchal@sunpharma.com
Arrows indicate
cone nuclei in
ONL.
Smaller, darker
nuclei are rod
nuclei.
Plastic-embedded
NHP retina –
1 µm section,
toluidine blue stain
Please email questions to
Satish.Panchal@sunpharma.com
Transmission electron micrograph – rod
outer segments - NHP
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Satish.Panchal@sunpharma.com
Immunohistochemistry
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Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
NHP - Mononuclear cell infiltrates in
ciliary body/choroid – common
background finding
NHP – Glial Nodule & Cystic Retinal
Degeneration at Ora Serrata
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Satish.Panchal@sunpharma.com
Light-Induced Retinal Degeneration –
Albino Rodents
NORMAL
Loss of photoreceptors.
Fewer nuclei in outer
nuclear layer.
AFFECTED
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Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Satish.Panchal@sunpharma.com
Another (rare) Spontaneous Finding - Outer
Retinal Degeneration in a Mauritius Cynomolgus
Monkey
Inner Nuclear Layer
Outer Nuclear Layer
Outer Plexiform Layer
RPE RPE
Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
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
Please email questions to
Satish.Panchal@sunpharma.com
NHP – Immune-Mediated Inflammation -
Intravitreal Humanized Monoclonal Ab –
Perivascular Cuffs of Leukocytes
Please email questions to
Satish.Panchal@sunpharma.com
Artifact
NHP – Immune-Mediated Inflammation -
Intravitreal Humanized Monoclonal Ab –
Vitreous Cells
Please email questions to
Satish.Panchal@sunpharma.com
Artifact
NHP – Immune-Mediated Inflammation -
Intravitreal Humanized MAb – Perivascular
Cuffs of Leukocytes – Optic Disc Please email questions to
Satish.Panchal@sunpharma.com
Fovea - unremarkable
Please email questions to
Satish.Panchal@sunpharma.com
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.
Please email questions to
Satish.Panchal@sunpharma.com
Multifocal necrosis of rabbit lacrimal gland
Please email questions to
Satish.Panchal@sunpharma.com
Necrosis of rabbit lacrimal gland
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Satish.Panchal@sunpharma.com
Necrosis of rabbit Harderian gland
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Satish.Panchal@sunpharma.com
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.
Please email questions to
Satish.Panchal@sunpharma.com
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.
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