ArticlePDF AvailableLiterature Review

The SPOTS System: An Ocular Scoring System Optimized for Use in Modern Preclinical Drug Development and Toxicology

Authors:

Abstract and Figures

Purpose: To present a semiquantitative ocular scoring system comprising elements and criteria that address many of the limitations associated with systems commonly used in preclinical studies, providing enhanced cross-species applicability and predictive value in modern ocular drug and device development. Methods: Revisions to the ocular scoring systems of McDonald-Shadduck and Hackett-McDonald were conducted by board-certified veterinary ophthalmologists at Ocular Services On Demand (OSOD) over the execution of hundreds of in vivo preclinical ocular drug and device development studies and general toxicological investigations. This semiquantitative preclinical ocular toxicology scoring (SPOTS) system was driven by limitations of previously published systems identified by our group's recent review of slit lamp-based scoring systems in clinical ophthalmology, toxicology, and vision science. Results: The SPOTS system provides scoring criteria for the anterior segment, posterior segment, and characterization of intravitreal test articles. Key elements include: standardized slit lamp settings; expansion of criteria to enhance applicability to nonrabbit species; refinement and disambiguation of scoring criteria for corneal opacity, fluorescein staining severity, and aqueous flare; introduction of novel criteria for scoring of aqueous and anterior vitreous cell; and introduction of criteria for findings observed with drugs/devices targeting the posterior segment. A modified Standardization of Uveitis Nomenclature (SUN) system is also introduced to facilitate accurate use of SUN's criteria in laboratory species. Conclusions: The SPOTS systems provide criteria that stand to enhance the applicability of semiquantitative scoring criteria to the full range of laboratory species, in the context of modern approaches to ocular therapeutics and drug delivery and drug and device development.
Content may be subject to copyright.
The SPOTS System:
An Ocular Scoring System Optimized for Use in Modern
Preclinical Drug Development and Toxicology
Joshua Seth Eaton,
1,2
Paul E. Miller,
1,3
Ellison Bentley,
1,3
Sara M. Thomasy,
1,2
and Christopher J. Murphy
1,2,4
Abstract
Purpose: To present a semiquantitative ocular scoring system comprising elements and criteria that address
many of the limitations associated with systems commonly used in preclinical studies, providing enhanced
cross-species applicability and predictive value in modern ocular drug and device development.
Methods: Revisions to the ocular scoring systems of McDonald–Shadduck and Hackett–McDonald were
conducted by board-certified veterinary ophthalmologists at Ocular Services On Demand (OSOD) over the
execution of hundreds of in vivo preclinical ocular drug and device development studies and general toxico-
logical investigations. This semiquantitative preclinical ocular toxicology scoring (SPOTS) system was driven
by limitations of previously published systems identified by our group’s recent review of slit lamp-based
scoring systems in clinical ophthalmology, toxicology, and vision science.
Results: The SPOTS system provides scoring criteria for the anterior segment, posterior segment, and char-
acterization of intravitreal test articles. Key elements include: standardized slit lamp settings; expansion of
criteria to enhance applicability to nonrabbit species; refinement and disambiguation of scoring criteria for
corneal opacity, fluorescein staining severity, and aqueous flare; introduction of novel criteria for scoring of
aqueous and anterior vitreous cell; and introduction of criteria for findings observed with drugs/devices tar-
geting the posterior segment. A modified Standardization of Uveitis Nomenclature (SUN) system is also
introduced to facilitate accurate use of SUN’s criteria in laboratory species.
Conclusions: The SPOTS systems provide criteria that stand to enhance the applicability of semiquantitative
scoring criteria to the full range of laboratory species, in the context of modern approaches to ocular thera-
peutics and drug delivery and drug and device development.
Keywords: slit lamp, scoring, semiquantitative, toxicology, preclinical
Introduction
Current slit lamp-based ocular scoring systems allow
examiners to semiquantitatively assess clinical findings
when evaluating the eye’s anterior segment. The Standar-
dization of Uveitis Nomenclature (SUN) system, for ex-
ample, was developed to provide a standardized, objective
method for assessing human patients with naturally occur-
ring uveitis, and is widely used in physician-based oph-
thalmology.
1
Descriptions and applications of slit lamp-
based scoring systems have also been reported for decades
in toxicological investigations involving nonhuman labora-
tory species.
2–4
In these species, semiquantitative systems
described by McDonald and Shadduck,
5
and Hackett and
McDonald
6
are frequently cited in the preclinical drug de-
velopment literature.
2–4
These systems and others were compared in our recent
comprehensive literature review of slit lamp-based scoring
systems in clinical ophthalmology, ocular toxicology, and vi-
sion science. That review identified 138 published systems for
assessment of the general anterior segment, lens, or ocular
surface. While most systems were described for the clinical
evaluation of human patients, 17% (including those of
McDonald–Shadduck and Hackett–McDonald) were described
1
Ocular Services On Demand (OSOD), LLC, Madison, Wisconsin.
2
Department of Surgical & Radiological Sciences, School of Veterinary Medicine, University of California—Davis, Davis, California.
3
Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin—Madison, Madison, Wisconsin.
4
Department of Ophthalmology & Vision Science, School of Medicine, University of California—Davis, Sacramento, California.
JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS
Volume 33, Number 10, 2017
ªMary Ann Liebert, Inc.
DOI: 10.1089/jop.2017.0108
718
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
specifically for albino rabbits, with 11% and 4% being re-
ported for evaluation of albino rats and mice, respectively.
Systems described for pigmented strains of rabbits and rats,
and larger species, such as dogs and nonhuman primates
(NHPs), were infrequently described.
Likely due to lack of available systems with strain- or
species-specific scoring criteria, there is an industry-wide
tendency to apply systems such as those of McDonald–
Shadduck and Hackett–McDonald (or modified versions
thereof) to pigmented strains as well as larger laboratory
species such as the dog and NHP. Importantly, these systems
do not incorporate standard slit lamp settings for examiners,
and use criteria that may not be completely congruous with
modern approaches to ocular drug delivery or development of
novel ocular therapeutics and devices. Additionally, the lack
of stringent application of uniform instrument settings and the
intrinsic ambiguities of applying systems to strains and spe-
cies they were not specifically designed for can be amplified
in extended studies that may necessitate exams being per-
formed by multiple examiners. To address many of these
limitations and improve upon current approaches to clinical
evaluation and semiquantitative scoring in the full range of
albino and pigmented species used in modern ocular drug
development, we present herein the semiquantitative pre-
clinical ocular toxicology scoring (SPOTS) system as well as
the SPOTS-modified SUN system.
The SPOTS System
The SPOTS system evolved from the iterative collaborative
engagement of a team of scoring-harmonized, board-certified
veterinary ophthalmologists, members of Ocular Services On
Demand (OSOD), over the execution of hundreds of ocular
drug and device development studies evaluating a wide array of
test articles and devices, material platforms, and delivery
routes. Harmonization was verified by the sequential scoring of
all board-certified veterinary ophthalmologists using the oph-
thalmologist with the most extensive experience in the context
of drug development studies (P.E.M.) as the gold standard in
application of the scoring system employed. It expands upon or
modifies many criteria employed in the systems of McDonald–
Shadduck and Hackett–McDonald, introduces novel anterior
chamber (AC) and vitreous cell scoring schemes, and provides
standardized slit lamp settings applicable to both table-
mounted and hand-held instruments. Beyond specific param-
eters scored using slit lamp biomicroscopy, the SPOTS system
also introduces ophthalmoscopic scoring criteria for posterior
segment parameters with importance in ocular toxicology, as
well as criteriafor accurate monitoringof intravitreal (IVT) test
articles. It should be noted that the SPOTS system utilizes
criteria for real-time, analog scoring by clinicians, and not
digital scoring systems or similar schemes that incorporate
grading center-level analysis or other techniques for objective
quantification as previously published.
7–13
An important aspect of the SPOTS system is the option for
integration of clinical diagrams to capture important attributes
of ocular findings or test articles that are not easily addressed
through scoring and tabulation, but which provide information
of considerable value in drug development programs. In ad-
dition, to accurately record the severity and spatial distribution
of ocular findings within the anterior and posterior segments,
diagrammatic representations improve correspondence of
findings across sequential observers. Furthermore, integration
of diagrams is especially useful when conducting eye-targeted
programs, but is typically left out (at least initially) in ocular
assessments of noneye targeted therapeutics.
It needs to be acknowledged that any system employed in
ocular drug or device development programs may benefit by
the integration of study-specific scoring parameters into the
basal scoring system being employed. While the SPOTS sys-
tem provides the default system used among all OSOD veter-
inary ophthalmologists and vision scientists, study-specific
pilot data, unanticipated clinical findings, or the specific needs
of a particular program may necessitate modifications. Based
on the limitations of currently published scoring systems,
however, the updated and novel elements of the SPOTS sys-
tem, whether implemented as published or modified, stand to
improve the predictive value of ocularscoring in the context of
modern drug development.
The SPOTS system comprises 3 sections: (1) anterior
segment and anterior vitreous cell scoring through slit lamp
biomicroscopy; (2) posterior segment scoring through indi-
rect ophthalmoscopy; and (3) IVT test article characterization
also primarily through indirect ophthalmoscopy. Complete
scoring criteria are presented in Table 1.
Below we detail the elements of this system, employed by
all members of OSOD’s distributed preclinical consulting
network. For simplicity, we highlight similarities and dis-
tinguishing attributes of the SPOTS system from the most
widely referenced systems detailed by McDonald–Shadduck
and Hackett–McDonald (M-S and H-M).
Section I. Anterior Segment
and Anterior Vitreous Cell Scoring
Anterior segment scoring is performed using a Kowa
SL-15 or SL-17 hand-held slit lamp or equivalent. In the
conduction of drug/device development programs, hand-
held slit lamps are generally preferred to table-mounted
instruments for the efficient evaluation of a large number of
laboratory animals which, with the exception of NHPs, are
typically unanesthetized. Furthermore, laboratory species
generally have skull conformations and behavioral traits that
are poorly suited to examination using fixed table-mounted
devices. Examinations are performed using high-beam il-
lumination (as tolerated) and a magnification level suitable
for identification of aqueous or vitreous cells in the species
being examined (typically 10 ·to16 ·). Diffuse illumination
is used to survey the adnexa and ocular surface and evaluate
the pupillary light reflex (PLR); and illumination with a slit
beam is used to critically evaluate the anterior segment
structures and anterior media (aqueous humor, lens, and
anterior vitreous). The slit beam is set to a default width and
height of 0.2 and 12 mm, respectively, with the beam angled
at *30–45to the eye’s vertical axial (90) meridian. Note
that at high magnification (eg, 16 ·), the examiner will not
visualize the full-beam height passing through the anterior
segment of the eye, unless the species being examined has a
vertical corneal diameter (VCD) £12 mm. In those species,
beam height is decreased accordingly.
Pupillary light reflex
Scoring of the PLR is similar to those of M-S and H-M,
but incorporates an additional score (‘‘3’’), specifying mi-
osis as observed with anterior uveitis, or as a class effect of
THE SPOTS SCORING SYSTEMS 719
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
Table 1. The Semiquantitative Preclinical Ocular Toxicology Scoring System
The SPOTS system includes scoring schemes and elements adapted from the anterior segment systems of McDonald–
Shadduck (M-S)
5
and Hackett–McDonald (H-M),
6
and the scoring scheme for vitreous haze of Nussenblatt et al.
28
For
many parameters, the schemes designated by the SPOTS system diverge from M-S to H-M to (1) enhance applicability to
multiple laboratory species; and (2) improve relevance to the routes, delivery systems, and approaches used in modern
ocular drug development programs.
The SPOTS system comprises 3 sections: (1) anterior segment and anterior vitreous cell scoring through slit lamp
biomicroscopy; (2) posterior segment scoring through indirect ophthalmoscopy; and (3) characterization of IVT test
articles primarily through ophthalmoscopy. The SPOTS-modified SUN system presents a scheme for scoring of AC cell
to mimic that of SUN,
1
and is presented at the end of this table.
SECTION I: ANTERIOR SEGMENT AND VITREOUS CELL
Scoring of the anterior segment is performed using a KowaSL-15 or SL-17 hand-held slit lamp or equivalent. A diffuse
beam setting is used to survey the adnexa and ocular surface, and for scoring of the PLR, conjunctival congestion,
conjunctival discharge, conjunctival swelling, iris involvement, and corneal opacity. Corneal fluorescein staining scoring
can be aided by integrating the cobalt blue filter into the diffuse light path. For scoring of lesions of the clear media,
including corneal opacity, corneal vascularization, AC flare, AC cell (total number of AC cells viewed in the slit beam),
lens opacity, and anterior vitreous cell, the slit beam is set at default beam height (12 mm), 0.2 mm beam width, with an
*30–45beam angle. The highest setting for illumination (as tolerated) and a magnification level suitable for
identification of aqueous or vitreous cells in the species being examined (typically 10 ·to16 ·) are set. It is noteworthy
that the examiner will not see the full beam height at 16 ·magnification, unless the species being examined has a VCD
£12 mm.
**Note that scoring of corneal opacity and fluorescein staining requires examiners to assign 2 distinct numerical scores.
Observation/lesion Score Description
PLR 0 Normal, brisk pupillary reflex.
1 Pupil is relatively dilated and/or dyscoric with a sluggish
pupillary reflex.
2 Pupil is fully dilated and/or dyscoric with no detectable
pupillary reflex.
3 Miotic pupil. PLR is difficult to evaluate, due to the
small pupil size.
Conjunctival hyperemia 0 Bulbar conjunctiva is normal in appearance for the species.
Small, pale pink vessels may be observed, primarily at, or
adjacent to, the limbus. In rabbits, small vessels associated
with the dorsal and ventral rectus muscles may be seen at
12 and 6 o’clock, respectively.
1 Pink-to-red bulbar conjunctival vessels with minimal branching
are visible extending 1–3 mm posteriorly from the limbus
toward the conjunctival fornix. In rabbits, pink-to-red
perilimbal vessels are visible primarily at or adjacent to
the upper (12 o’clock) and lower (6 o’clock) aspects of
the perilimbal region.
2 Prominent red bulbar conjunctival vessels with multiple
branches are visible extending from the limbus to the
conjunctival fornix. In rabbits, prominent red perilimbal
vessels are visible involving at least 75% of the circumference
of the perilimbal region.
3 Red-to-dark red, engorged bulbar conjunctival vessels with
extensive branching and/or tortuosity are visible extending
from the limbus to the conjunctival fornix. The conjunctiva
between large vessels may have a flushed pink-to-red
appearance. In rabbits, red-to-dark red engorged perilimbal
vessels are visible, with pink-to-red flushing of the adjacent
bulbar conjunctiva. In any species, bulbar and/or palpebral
conjunctival petechia may be present.
Conjunctival
swelling (chemosis)
0 No abnormal swelling of the conjunctival tissue.
1 Bulbar conjunctival swelling above normal is observed focally,
most commonly in the perilimbal region with no eversion
of the eyelid(s) or change in eyelid margin contour.
2 Bulbar conjunctival swelling above normal is observed
diffusely, but with no eversion of the eyelid(s) or change
in eyelid margin contour.
(continued)
720
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
Table 1. (Continued)
Observation/lesion Score Description
3 Bulbar and palpebral conjunctival swelling is observed,
resulting in eyelid eversion and/or misalignment. The
eyelid margin(s) may appear swollen and/or have an irregular,
‘‘undulating’’ contour, but can still be closed completely.
4
a
Bulbar and palpebral conjunctival swelling is severe, partially
or completely obscuring examination of the rest of the
ocular surface and globe. Eyelid closure is incomplete
with exposed bulbar and/or palpebral conjunctiva protruding
between the eyelid margins.
Conjunctival discharge 0 No abnormal discharge.
1 Discharge is above normal and present on the surface of the eye
or in the medial canthus, but not on the lids or hairs
of the eyelids.
2 Discharge is abundant, easily observed, and has accumulated
on the lids and around the hairs of the eyelids.
3 Discharge has been flowing over the eyelids so as to wet or
accumulate on the hairs of the eyelids and the skin around
the eyes, past the orbital rim.
**For corneal opacity, 2 scores are assigned; 1 for corneal opacity (severity), and 1 for corneal opacity (area)
Corneal opacity (severity)
b
0 Normal cornea. Appears with the slit lamp as having a bright
gray line on the epithelial surface and a bright gray line on
the endothelial surface with a marble-like gray appearance
of the stroma.
1 Minimal loss of corneal transparency. With diffuse
illumination, the underlying anterior segment structures
are clearly visible, although corneal opacity is apparent to
an experienced observer.
2 Mild loss of corneal transparency. With diffuse illumination, the
underlying anterior segment structures are visible, although
there is a reduction in the ability to appreciate their detail.
3 Moderate loss of corneal transparency. With diffuse illumination,
there is a greater inability to see the details of the underlying
anterior segment structures than with a score of 2, but the
observer is still able to score aqueous flare, iris vessel
congestion, observe for pupillary response, and note
lenticular changes.
4 Severe loss of corneal transparency. With diffuse illumination,
the underlying anterior segment structures cannot be seen
so that the evaluation of aqueous flare, iris vessel congestion,
pupillary response, and lenticular changes is not possible.
Corneal opacity (area)
c
0 Normal cornea with no area of corneal opacity.
1 1% to 25% area of corneal opacity.
2 26% to 50% area of corneal opacity.
3 51% to 75% area of corneal opacity.
4 76% to 100% area of corneal opacity.
Corneal vascularization 0 No corneal vascularization.
1 Vascularization is present but vessels have not penetrated
beyond 2 mm axially.
2 Vascularization is present and vessels have penetrated
2 mm axially.
AC flare 0 No protein is visible in the AC when viewed by an experienced
observer using slit lamp biomicroscopy, a small, bright,
focal slit beam of white light, and high magnification.
0.5 (trace) Trace amount of protein is detectable in the AC. This protein
is only visible with careful scrutiny by an experienced observer
using slit lamp biomicroscopy, a small, bright, focal slit
beam of white light, and high magnification.
(continued)
721
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
Table 1. (Continued)
Observation/lesion Score Description
1 Mild amount of protein is detectable in the AC. The presence
of protein in the AC is immediately apparent to an
experienced observer using slit lamp biomicroscopy and high
magnification, but such protein is detected only with careful
observation with the naked eye and a small, bright, focal slit
beam of white light.
2–3 Moderate amount of protein is detectable in the AC. These
scores are similar to Score 1, but the opacity would be readily
visible to the naked eye of an observer using any source
of a focused beam of white light. This is a continuum of
moderate opacification, with Score 2 less apparent
than Score 3.
4 Large (severe) amount of protein is detectable in the AC.
Similar to Score 3, but the optical density of the protein
approaches that of the lens behind it. Additionally, frank
fibrin deposition is frequently seen in acute circumstances.
It needs to be noted that because fibrin may persist
for a period of time after partial or complete restoration of the
blood–aqueous barrier, it is possible to have resorbing fibrin
present with lower numeric assignations for flare (eg, Score 1
flare with fibrin).
Total number of AC cells viewed in volume (field) of the slit beam*
0 No cells in field.
0.5 (trace) 1–5 cells in field.
1 6–25 cells in field.
2 26–50 cells in field.
3 51–100 cells in field.
4>100 cells in field.
*All AC cell scores should be accompanied by one of the following notations of cell color: predominantly (*75%) white,
mixed color (*50% white, 50% brown and/or red), predominantly (*75%) brown, or predominantly (*75%) red.
Iris involvement
d
0 Normal iris without any hyperemia of the iris vessels.
1 Minimal injection of secondary vessels but not tertiary.
2 Minimal injection of the tertiary vessels and
minimal-to-moderate injection of the secondary vessels.
3 Moderate injection of the secondary and tertiary vessels
with a slight swelling of the iris stroma (this gives the
iris surface a slightly rugose appearance).
4 Severe injection of the secondary and tertiary vessels with
marked swelling of the iris stroma. The iris appears
rugose; may be accompanied by hemorrhage in the
iris or AC (hyphema).
Anterior vitreous cell* 0 £1 cell in field.
0.5 (trace) 1–5 cells in field.
1 6–25 cells in field.
2 26–50 cells in field.
3 51–100 cells in field.
4>100 cells in field.
*All anterior vitreous cell scores should be accompanied by one of the following notations of cell color: predominantly
(*75%) white, mixed color (*50% white, 50% brown and/or red), predominantly (*75%) brown, or predominantly
(*75%) red.
**For fluorescein staining 2 scores are assigned; 1 for fluorescein staining (severity), and 1 for fluorescein
staining (area)
Fluorescein staining (severity)
e
0 Absence of fluorescein staining.
1 Slight multifocal punctate fluorescein staining.
2 Distinct multifocal to coalescent punctate fluorescein staining.
3 Frank fluorescein staining associated with focal or
multifocal epithelial loss, but no stromal loss.
4 Frank fluorescein staining associated with focal
or multifocal epithelial and stromal loss.
Fluorescein staining (area)
f
0 No area of fluorescein staining.
1 1% to 25% area of fluorescein staining.
2 26% to 50% area of fluorescein staining.
3 51% to 75% area of fluorescein staining.
4 76% to 100% area of fluorescein staining.
(continued)
722
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
Table 1. (Continued)
Observation/lesion Score Description
Lens opacity
g
The lens should be evaluated routinely during ocular evaluations and scored as 0 (normal) or 1 (abnormal).
The presence of lenticular opacities should be described and the location noted as defined below.
The crystalline lens is readily observed with the aid of the slit lamp biomicroscope, and the location of lenticular
opacity can readily be discerned by direct and retroillumination. The location of lenticular opacities can be
arbitrarily divided into the following lenticular regions beginning with the anterior capsule:
Anterior capsule Anterior cortical Nuclear Posterior subcapsular
Anterior subcapsular Equatorial cortical Posterior cortical Posterior capsule
Punctate: A focal or multifocal, discrete, dot-like lens opacity that is visible only to a trained observer with a SL-15 or
SL-17 slit lamp biomicroscope (or equivalent) at 10 ·to 16 ·magnification.
Incipient: A focal lens opacity that is visible upon gross inspection of the eye with an indirect ophthalmoscope or other
focal light source and retroillumination. The view of the fundus is minimally impaired by the opacity. Upon slit lamp
biomicroscopy, the opacity can be localized to a specific region of the lens and other regions of the lens appear normal.
Incomplete: A diffuse lens opacity visible upon gross inspection of the eye with an indirect ophthalmoscope or other focal
light source and retroillumination. The view of the fundus is significantly impaired, but a fundus reflex can still be
obtained. Upon slit lamp biomicroscopy the opacity involves multiple regions of the lens.
Complete: A diffuse lens opacity visible upon gross inspection of the eye with an indirect ophthalmoscope or other focal
light source. The fundus cannot be seen and a fundus reflex cannot be elicited. Upon slit lamp biomicroscopy the entire
lens is opaque.
Resorbing: A diffuse lens opacity visible upon gross inspection of the eye with an indirect ophthalmoscope or other focal
light source. The fundus may or may not be visible and a fundus reflex may or may not be elicited. The lens capsule may
be wrinkled and the lens itself is dehydrated and flattened or liquid and soft in appearance. Upon slit lamp biomicroscopy
the entire lens is involved in the opacity.
a
Conjunctival swelling scored as ‘‘4’’ may present with varying degrees of severity, primarily depending on the degree of conjunctival
exposure.
b
If multiple areas of opacity with varying scores are observed in an eye, the assigned Severity score should correspond to the most
severely affected area.
c
If multiple corneal opacities are observed in an eye, diagramming of the lesions’ characteristics is recommended in lieu of a corneal
opacity (area) score.
d
In pigmented strains/species, visibility of secondary and tertiary iridal vessels may be obscured.
e
If multiple fluorescein stain severity scores are observed in an eye, the assigned Severity score should correspond to the most severely
affected area.
f
If multiple fluorescein staining lesions are observed in an eye, diagramming of staining distribution is recommended in lieu of a
fluorescein staining (area) score.
g
Diagramming of lens opacities is recommended to facilitate localization and longitudinal monitoring.
AC, anterior chamber; IVT, intravitreal; PLR, pupillary light reflex; SPOTS, semi-quantitative preclinical ocular toxicology scoring;
SUN, Standardization of Uveitis Nomenclature; VCD, vertical corneal diameter.
SECTION II: POSTERIOR SEGMENT
Scoring of the posterior segment is performed using indirect ophthalmoscopy with an adjustable ophthalmoscopic headset
and condensing lens. Dioptric strength of the condensing lens is at the discretion of the examiner. Recommended dioptric
strengths for different laboratory species are as follows: Volk 2.2 PanRetinal, 14D, or 20D for nonhuman primates; 28D
for rabbits, pigs, and dogs; 40D for rats; and 60D for mice.
Parameters scored in the posterior segment include: vitreous haze (modified by the system of Nussenblatt et al.
28
), degraded
fundus view, and retinal perivascular sheathing (commonly observed in nonhuman primates but also in dogs and rats).
Observation/lesion Score Description
Vitreous haze 0 No vitreous haze.
0.5 Minimal vitreous haze with only slight blurring of the margins of the
optic disk and retinal vasculature.
1 Mild vitreous haze with slight blurring of the margins of the optic
disk. View of the retinal vasculature is partially obscured, but
vessels are still visible from the disk to the periphery.
2 Moderate vitreous haze with blurring of the optic disk margins. The
retinal vasculature is nearly completely obscured.
3 Marked vitreous haze in which the optic disk can be viewed, but is
nearly obscured; and the retinal vasculature is completely
obscured.
4 Severe vitreous haze in which the details of all structures, including
the optic disk, are obscured.
(continued)
723
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
certain drugs (eg, topical prostaglandin analogs or pilocar-
pine in dogs), complicating evaluation of pupil function.
Dyscoria (misshapen pupil) is also incorporated into scoring
criteria to include both functional and structural pupil ab-
normalities (eg, postinflammatory posterior synechia) as
causes for slow or absent PLR.
Conjunctiva
Scoring divisions for the conjunctiva (eg, congestion,
swelling, and discharge) are identical to those of M-S and H-
M. However, previous criteria described for scoring of ocular
irritation were largely developed to accommodate toxicity
evaluation of nonpharmaceutical products. Furthermore, con-
junctival schemes in those systems were specifically developed
for recording observations in albino rabbits. In the case of
conjunctival hyperemia (synonymous with ‘‘conjunctival
congestion’’), this is problematic since the rabbit’s primary
conjunctival vasculature follows a distinctly perilimbal ana-
tomical distribution, producing clinical patterns of congestion
whose characteristic features are not necessarily shared by
other species. Therefore, SPOTS criteria for conjunctival hy-
peremia and swelling (chemosis) in rabbits have been slightly
modified to more accurately denote features of irritation and
reactivity commonly observed in modern ocular drug and
device development. Additionally, new criteria have been ad-
ded to describe patterns of conjunctival hyperemia applicable
to nonrabbit species (Table 1 and Fig. 1). Scoring criteria for
Table 1. (Continued)
Observation/lesion Score Description
Degraded fundus view 0 Normal, unobstructed fundus view.
1 Mildly degraded view. Details of fundus anatomy are visible but
slightly dulled.
2 Moderately degraded view. Normal features of fundus anatomy can
be identified, but details cannot be discerned.
3 Severely degraded view. Normal features of fundus anatomy cannot
be identified.
Retinal perivascular sheathing
a
0 Normal. No perivascular sheathing.
1 Mild perivascular sheathing. Focal perivascular haze/infiltrate is
visible, but faint/barely discernible and confined to 1 quadrant of
the fundus when viewed with an indirect ophthalmoscope.
2 Moderate perivascular sheathing. White perivascular haze/infiltrate is
clearly visible and involves multiple vessels in >1 fundus quadrant.
3 Severe perivascular sheathing. Dense white perivascular infiltrate is
visible (often resembling human frosted branch angiitis
32,33
) with
segmental or confluent distribution involving most vessels of the
fundus. In some cases, hemorrhage may accompany severe
perivascular sheathing.
a
Diagramming of retinal perivascular sheathing is recommended to facilitate clinical appearance, distribution, and longitudinal
monitoring.
SECTION III: IVT TEST ARTICLE
Scoring for IVT test articles may be indicated to: (1) verify its successful delivery following dosing; (2) characterize its
appearance, location, or distribution longitudinally following injection or implantation; (3) identify and document its
degradation; and (4) to facilitate identification and interpretation of associated adverse effects. Scoring of IVT test articles
(eg, solutions, suspensions, IVT implants/devices, microparticle systems) is performed using indirect ophthalmoscopy,
but evaluation of test article in the anterior vitreous or AC can be supplemented using slit lamp biomicroscopy.
Test article scoring requires the examiner to assign 2 numerical scores: (1) test article appearance and (2) test article
distribution.
Observation/lesion Score Description
Test article appearance 1 The test article is transparent, but may create refractive shift when
viewing underlying fundus structures.
2 The test article is translucent, permitting only a hazy view of
underlying fundus structures.
3 The test article is opaque, obscuring view of underlying fundus
structures.
Test article distribution
a
1 Test article is a focal mass/depot, or an intact solid implant.
2 Test article is a focal mass/depot with adjacent strands/wisps, or the
implant has fragmented.
3 Test article is a focal mass/depot with adjacent strands/wisps and
some diffusion.
4 Test article is a focal mass/depot with some diffusion.
5 Test article is dispersed throughout vitreous.
a
Diagramming of test article distribution is recommended to facilitate characterization, localization, and longitudinal monitoring.
724 EATON ET AL.
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
conjunctival discharge remain largely unchanged, but a score
of3hasbeenmodiedtospecifythepresenceofdischarge
beyond the orbital rim, providing an anatomical landmark to
more clearly distinguish it from an adjacent score of 2.
Corneal opacity and fluorescein staining
As in the systems of M-S and H-M, a corneal opacity [any
lesions(s) reducing normal corneal transparency] and fluo-
rescein staining are each assigned two scores, one for se-
verity and the other for area affected. However, previous
criteria describing severity of these lesions are complex,
incorporating multiple descriptive features for an individual
score. For example, individual scoring criteria for corneal
opacity severity in the M-S and H-M systems differentiate
scores based on both increasing loss of transparency and
anterior to posterior (A-P) lesion distribution within the
cornea. Similarly, criteria for fluorescein staining severity
differentiate scores based on both description of increasing
staining intensity as well as distribution. For both parame-
ters, however, the features comprising each score may not
always be clinically observed as described, complicating the
examiner’s ability to identify and ascribe a single score.
Furthermore, the A-P distribution features described for
corneal opacities in these systems, while often applicable in
the evaluation of topically applied substances or drugs, may
not correlate with the range of ocular responses observed
following intraocular injections or other modern routes of
administration. It must also be noted that opacity severity
may vary across a single cornea depending on the route of
drug administration or location of an intraocular device,
features not accounted for in previously published systems.
The SPOTS scheme for corneal opacity severity is de-
fined solely by opacity density (loss of transparency and
obscuration of underlying anterior segment structures)
without consideration of depth or distribution within the
cornea. This simplifies and improves clarity of the clinical
observations and, in our group’s experience, promotes im-
proved consistency across observers in a single study. For
capture and documentation of other important lesion fea-
tures such as stromal distribution, association with altered
corneal thickness, or lesion color (eg, blue discoloration
with corneal edema), the examiner should incorporate a
clinical diagram. An additional option is to enhance the
information contained in diagrams of any anterior segment
finding(s) by incorporating standard color codes as previ-
ously published
14
(Fig. 2).
For fluorescein staining severity, SPOTS schemes corre-
spond to increasing severity of corneal surface disease and/
or ulceration, ranging from slight to mild corneal epithelial
damage/loss of intercellular integrity, to complete epithelial
loss, and finally to both epithelial and stromal loss. Com-
pared with M-S and H-M, this scheme provides clearer
distinction between scores, and in the context of topically
applied ophthalmic drugs, better distinguishes more minor
clinical corneal epithelial toxicity (Score £‘‘2,’’ Fig. 3) from
more severe forms. As for corneal opacity, diagramming of
stain retention is recommended to provide information about
its distribution, intensity, or other characteristics (Fig. 2).
The assigned severity score for corneal opacity or fluo-
rescein staining should correspond to the most severely af-
fected area (the area with the highest score), accompanied by
a diagram to characterize the distribution of multiple lesions.
For both corneal opacity and fluorescein staining, scoring
schemes for lesion area are identical to M-S and H-M.
Corneal vascularization (pannus)
Corneal vascularization schemes are slightly modified
compared with M-S and H-M. It is noteworthy that the term
‘‘pannus’’ bears different clinical meanings in physician-
based and veterinary ophthalmology. While in physician-
based ophthalmic practice, pannus refers to any nonspecific
vascular infiltration of the cornea, canine pannus is
FIG. 1. (A, B) Photographic exemplars demonstrating conjunctival hyperemia scoring criteria using the SPOTS system in
(A) a nonrabbit species (canine images presented) and (B) rabbits (albino strain images presented). Note the more radial
distribution of hyperemic conjunctival vessels with extension toward the conjunctival fornix in the canine images, and the
primarily perilimbal distribution of hyperemic conjunctival vessels in the rabbit images. SPOTS, semiquantitative pre-
clinical ocular toxicology scoring.
THE SPOTS SCORING SYSTEMS 725
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
frequently used in veterinary ophthalmology to identify a
specific immune-mediated disease (also known as chronic
superficial keratitis or U
¨berreiter’s syndrome), characterized
by progressive fibrovascular, lymphocytic, and plasmacytic
infiltration of the cornea.
15,16
In the context of preclinical
drug and device development programs, the more nonspecific
definition of pannus, as employed by physicians, is more
appropriate for lesion description. The M-S and H-M systems
differentiated vascularization scores using criteria that in-
cluded descriptions of both vessel length and distribution. As
with corneal opacity and fluorescein staining severity scoring,
however, the features of both may not always be observed
together in the clinical patterns described. Thus, the SPOTS
system scores corneal vascularization solely on the basis of
vessel length to reduce ambiguity.
Aqueous (AC) flare
The systems of M-S and H-M designated 4 scores for
aqueous flare (AC protein) ranging from ‘‘0’’ (no flare) to
‘‘3,’’ based on increasing visibility of a Tyndall effect
within the AC as well as the apparent density of flare rela-
tive to the optical density of the adjacent lens. The SUN
FIG. 2. Example of corneal and anterior segment lesion diagramming using a modified standardized color-coding
system.
14
FIG. 3. Photographic exemplar demonstrating criteria
characteristic of a score ‘‘2’’ for fluorescein staining severity
using the SPOTS system (NHP image presented). NHP,
nonhuman primate.
726 EATON ET AL.
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
system designates 5 scores for flare, ranging from ‘‘0’’ (no
flare), to ‘‘4’’ [intense (fibrin or plastic)], with scores cor-
responding to faint, moderate, and marked flare in between.
Like M-S and H-M, the SPOTS system utilizes the optical
density of the lens as a scoring reference. In the collective
authors’ experience, however, flare exceeding the optical
density of the lens is rarely observed in laboratory animals
in toxicology studies, even those with severe anterior uve-
itis; thus, a maximal aqueous flare score (‘‘4’’) is defined as
‘‘approaching the optical density of the lens.’’ Lesser scores
are defined in a similar manner to those described in M-S
and H-M and based upon Tyndall visibility in the AC, range
from mild (‘‘1’’) to moderate (‘‘2–3’’). In addition, the
SPOTS system incorporates a sixth score (‘‘0.5’’ or
‘‘trace’’), describing low-grade Tyndall effect visible only
with careful scrutiny by a highly experienced examiner
using a slit lamp. Severity of aqueous flare is a hallmark
feature of breakdown of the blood–aqueous barrier due to
inflammation or test article effect, so the SPOTS scheme
enhances the identification of often subtle differences in
aqueous flare associated with different test articles and their
formulations. The SPOTS system also clarifies that the
presence of AC fibrin does not unequivocally correlate to a
maximal flare score as indicated by SUN, as resorbing fibrin
can be observed in tandem with lower scores for flare.
Furthermore, it is recommended that fibrin or aqueous cel-
lular constituents (eg, hyphema, hypopyon) be diagrammed
by the examiner (Fig. 2) to not only capture their extent but
to also facilitate longitudinal monitoring.
Aqueous (AC) cell
Like aqueous flare, the presence and severity of aqueous
cell is a critical marker of anterior segment inflammation in
humans
17,18
and animals
4,19–21
; and scoring of this primary
inflammatory response is critical in safety and efficacy
evaluations of modern ophthalmic pharmaceuticals and de-
vices. Modern routes of administration include topical routes
and direct delivery of the test article (in solution, suspension,
or integrated into a variety of biomaterial platforms) into the
subconjunctival, AC, vitreous, suprachoroidal, and subretinal
spaces.
22,23
Aqueous cell is absent from the majority of
scoring schemes developed for laboratory animals, including
those of M-S and H-M.
2,4
The SUN system’s scheme for AC
cell scoring in human patients is frequently adopted for
scoring in laboratory animals, but the predictive value SUN
provides to monitoring human patients is more limited in
laboratory animals by species-related differences in ocular
anatomy and ocular reactivity.
24,25
It needs to be noted that species-related differences in
anterior segment geometry (eg, corneal curvature, AC depth,
anterior lens curvature) heavily influence the volume (and
percentage of the total AC volume) sampled by a slit beam
as documented in a recently published analysis (Fig. 4).
24
In
species with larger AC dimensions, the volume of aqueous
sampled by the slit beam can be up to 100% greater than
those of humans. Similarly, dramatically smaller aqueous
volumes are sampled in small-eyed animals such as ro-
dents.
24
Acknowledging the results of this analysis as well as
established species-related differences in ocular reactivity,
25
the AC cell value corresponding to SUN’s highest score for
human patients often prematurely tops out when used to
quantify AC cells in drug safety evaluations using labora-
tory species. Therefore, the SPOTS system uses compara-
tively higher cell numbers for scores ‘‘1’’ through ‘‘4.’’ This
enables examiners to better stratify anterior inflammatory
responses between different drug formulations or when
evaluating for dose-related inflammatory responses, across
the entire spectrum of laboratory species.
In addition to assigning a score for AC cell number, ex-
aminers should always indicate the predominant cell color
as observed using the slit lamp. Documentation of the pre-
dominant cell color may provide information as to their
FIG. 4. Comparative optical
coherence tomography (OCT)
images of comparative axial
cross-sectional anterior seg-
ment geometry in the human
(a), cynomolgus macaque (b),
dog (c), rabbit (d),rat(e),and
mouse (f). Reprinted from:
Thomasy et al.
24
THE SPOTS SCORING SYSTEMS 727
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
source and type (eg, infiltrating inflammatory cells, eryth-
rocytes, or melanocytes liberated from the anterior or pos-
terior uvea). Cell color is noted as mixed color or as
predominantly white, predominantly brown, or predomi-
nantly red, with a color being considered predominant when
it accounts for *75% of cells observed. In pigmented ani-
mals, these data can be useful in distinguishing between
observation of cells associated with the test article or the
procedure used to introduce it. With the latter, cells in the
AC are typically transient and predominantly white in color;
and in the vitreous, cell color is more commonly brown or
mixed and only observed at minimal (trace) levels. The
nature of these observations, however, can vary depending
on other characteristics of the injection procedure, the test
article, and individual animal variability.
Iris involvement
The SPOTS scheme for iris involvement (synonymous
with iris congestion or iridal vessel injection) is similar to
M-S and H-M, ranging from ‘‘0’’ to ‘‘4’’ depending on the
severity of vessel injection, and the presence or absence of
other structural features such as iris swelling or rugosity.
However, M-S and H-M also include descriptions of vas-
cular features specific to albino rabbits. For example, both
systems indicate that the normal iris (score of ‘‘0’’) may
include secondary and tertiary vessel hyperemia ‘‘occa-
sionally around the 12:00 to 1:00 o’clock position near the
pupillary border and the 6:00 to 7:00 o’clock position near
the pupillary border.’’ The terminology in the SPOTS sys-
tem, therefore, has been reduced to refine scoring criteria to
descriptions of vessel involvement and iris swelling only
(Table 1 and Fig. 5). It needs to be noted that identification of
secondary and tertiary iris vessels is difficult to impossible in
pigmented strains or species, and may require species/strain-
specific modifications to the scoring system.
Anterior vitreous cell
The slit lamp biomicroscope can be used to evaluate the
anterior vitreous for opacities or inflammatory cells. Slit
lamp-based vitreous cell scoring schemes, however, are not
widely described in the preclinical literature, despite being
commonly observed in eyes of animals undergoing intra-
ocular drug or device delivery (particularly after adminis-
tration to the posterior segment). The SPOTS system
incorporates a scheme for vitreous cell scoring, employing
the same semiquantitative criteria used for scoring of AC
cells (ranging from ‘‘0’’ to ‘‘4’’). Cell color is also noted
using the same descriptive terminology as for AC cells.
Lens opacity
Lens opacities are scored according to a scheme similar to
that of H-M, differentiated according to location within the
lens (eg, anterior or posterior capsular or cortical, nuclear,
equatorial, etc.). In addition, the SPOTS scheme recom-
mends a description of lens opacity size, ranging from
punctate to complete or resorbing. Diagramming of lens
opacities, especially when multiple lesions are observed, is
highly recommended to facilitate longitudinal monitoring
and interpretation.
FIG. 5. Photographic ex-
emplars demonstrating iris in-
volvement scoring criteria
using the SPOTS system (albi-
no rabbit strain images pre-
sented). Notes: No exemplar is
presented for Score ‘‘2’’; and
the conjunctival swelling and/
or congestion seen in images
representing Scores ‘‘3’’ and
‘‘4’’ are findings often observed
in association with moderate to
severe intraocular inflamma-
tion, though incidence and
severity may differ depending
on study-related factors or
individual animal variability.
728 EATON ET AL.
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
Section II. Posterior Segment Scoring
Unlike scoring for the anterior segment, the SPOTS
system does not specify instrument settings for examination
and scoring of the posterior segment. Nonetheless, scoring
of the posterior segment is most effectively (and efficiently)
performed using indirect ophthalmoscopy with an adjustable
headset and condensing lens. Clinical examiners at OSOD
recommend beginning with a Volk 2.2 PanRetinal, 14D,
or 20D lens for NHPs; a 28D lens for dogs, pigs, and rabbits;
a 40D lens for rats; and a 60D lens for mice. If a lesion is
observed, additional scrutiny is recommended using indirect
lenses of lower dioptric power for increased magnification
[including a 14D or 5.5D (macular) lens in larger-eyed
species], as well considering the use of a Welch Allyn
PanOpticophthalmoscope and/or direct ophthalmoscope
(or equivalent). When interpreting the characteristics of a
lesion(s), it should be noted that the actual lateral and axial
magnification perceived by the observer is directly influ-
enced by the dimensions of the globe being examined.
26
The
lower inherent magnification of larger eyes, for example,
will topographically flatten a lesion’s ophthalmoscopic ap-
pearance; therefore, this lesion would be attributed greater
clinical significance compared with the same lesion in a
small eye with higher magnification.
In addition to the use of numeric assignments, documen-
tation of posterior segment lesions should be complimented
using a clinical diagram. Similar to drawings of the anterior
segment, valuable additional information can be captured
through incorporation of diagrams using published standard
color coding schemes.
27
In our collective experience in ex-
amining subretinal dosing sites with a variety of test articles,
OSOD members have routinely incorporated diagrams in
tandem with ocular scoring to characterize their ophthalmo-
scopic appearance, spatial distribution, and degree of retinal
elevation. Together with diagramming of retinal lesions en
face, diagramming of lesions or IVT test articles in sagittal
drawings is also recommended to accurately record lesion/
test article shape, size, and location within the vitreous, fa-
cilitating longitudinal comparison and monitoring (Fig. 6).
FIG. 6. Example of posterior segment lesion diagramming using a modified standardized color-coding system.
27
The en
face diagram here presents lesions in the context of anatomical features of the NHP fundus (eg, holangiotic retinal vascular
pattern and macula). Lesions depicted comprise those typically observed in preclinical studies involving the posterior
segment, including the grid of macular/perimacular laser-induced lesions observed in animal models of choroidal
neovascularization.
41–44
Diagrammatic features may vary depending on the anatomical variations and specializations in
fundus anatomy of the species being examined (eg, merangiotic vascular pattern in rabbits, presence of a tapetum lucidum
in dogs, and lack of a true macula in most laboratory species). While green is conventionally used by vitreoretinal
physicians and surgeons to represent vitreous hemorrhage, our system has adopted red instead to disambiguate docu-
mentation of the lesion in different laboratory species. [D =dorsal (interchangeable with superior), V =ventral (inter-
changeable with inferior), T =temporal, N =nasal].
THE SPOTS SCORING SYSTEMS 729
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
When diagramming the posterior segment, examiners must
also remember that all features and lesions observed using
indirect ophthalmoscopy are reversed and inverted and take
care to accurately record/illustrate findings in their anatomi-
cally correct orientation.
Vitreous haze
Vitreous haze can be a useful indicator of posterior seg-
ment inflammation, as observed by Nussenblatt et al. at the
National Eye Institute (NEI) when they described photo-
graphic standards for scoring in human patients.
28
This
finding is also observed in association with posterior seg-
ment inflammation in laboratory species.
29–31
The SPOTS
system adopts and slightly modifies the NEI criteria with 6
scores ranging from ‘‘0’’ to ‘‘4’’ (including ‘‘0.5’’ or
‘‘trace’’), corresponding to increasing degree of blurring and
obscuration of the optic disk and retinal vasculature (Fig. 7).
Degraded fundus view
Uncommonly scored by published systems for laboratory
species, degradation of fundus view indicates general loss of
visible detail when examining the posterior segment with
scores ranging from ‘‘0’’ to ‘‘3,’’ corresponding to in-
creasing obscuration of details of fundus anatomy. Unlike
vitreous haze scoring whose criteria indicate an abnormality
confined to the posterior segment, however, any lesion(s) of
the optical pathway may contribute to the development of
degraded fundus view, including corneal opacification, AC
cells and/or flare, AC fibrin, lens opacity, vitreous haze,
vitreous floaters, IVT test articles, or any combination
thereof. Therefore, scoring of this parameter is not specific
for 1 clinical finding, but is more useful as an ‘‘area under
the curve’’ indicator of adverse events that affect clear vi-
sualization of the retina using ophthalmoscopy. It also
serves as a useful guide for gauging the veracity of clinical
observations made of posterior segment findings and can
assist in reconciling differences that may be found between
ophthalmoscopic and histopathological findings in the
course of a study.
Retinal perivascular sheathing
Perivascular sheathing (also referred to as ‘‘cuffing’’)
describes the presence of hazy to dense-white infiltrates
surrounding segments of retinal arterioles and/or venules,
and is unequivocally indicative of posterior segment in-
flammation (Fig. 8). It can vary in severity and can be
transient or progressive with sequential exposures to test
articles. The SPOTS system describes scores ranging from
‘‘0’’ to ‘‘3,’’ with descriptive criteria corresponding to
density of sheathing as well as area affected, as these 2
parameters typically correspond as severity increases. The
clinical appearance of human frosted branch angiitis
32,33
is
an excellent exemplar of this finding, illustrating the fea-
tures of moderate to severe perivascular sheathing that can
be observed in laboratory species in drug development
FIG. 7. Photographic images simulating vitreous haze scoring criteria using the SPOTS system (NHP fundus images
presented).
730 EATON ET AL.
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
programs. We have found scoring for this finding to be
particularly useful in the assessment of biologics delivered
through the IVT route.
Section III: IVT Test Article Scoring
IVT delivery is a popular modern route for delivery of
drugs or devices intended to treat the eye’s posterior seg-
ment, with a growing body of research supporting the de-
velopment of novel IVT delivery systems and material
platforms to optimize and sustain the delivery of therapeu-
tics in patients with degenerative retinal diseases.
22
Semi-
quantitative scoring and diagrammatic documentation of
these test articles, therefore, can provide valuable data re-
garding the behavior and fate of these agents as they can
disperse, migrate and/or dissolve, and in some cases, have
the potential for interfering with vision. Furthermore, mi-
gration into the AC presents the possibility for anterior
segment toxicity and/or impairing aqueous outflow. Clinical
characterization of IVT test articles is most commonly
achieved using indirect ophthalmoscopy, although slit lamp
biomicroscopy can complement assessment of test articles
in the anterior vitreous. While fundus photography can be
used as a complimentary endpoint, clinical scoring should
be based upon examination characteristics observed three
dimensionally.
IVT test article scoring requires the examiner to assign up
to 2 numerical scores to describe a test article’s appearance
and distribution. Scores for appearance correspond to the
general opacity of the test article, whereas distribution
scores describe a range of appearances corresponding to the
test article’s shape and profile on ophthalmoscopic obser-
vation. Diagramming of test article appearance, location,
and distribution is important as it facilitates longitudinal
monitoring, documentation of test article behavior in vivo,
and interpretation of accompanying examination findings.
The SPOTS-Modified SUN Scoring System
for AC Cells
The SUN System was originally described in 2005 to
improve and standardize the diagnostic terminology and
scoring schemes used in the evaluation of human patients
with naturally occurring uveitis (Table 2).
1
As previously
published systems for laboratory animals did not include
schemes for AC cells, SUN’s scoring scheme has become
increasingly applied to studies involving laboratory ani-
mals.
3
Furthermore, the use of a common system between
preclinical studies and human clinical trials offers the
promise of enhanced translatability of preclinical drug
safety evaluations.
The SUN scoring scheme for AC cells and aqueous flare
specifies use of a 1 ·1 mm slit beam, easily achieved on
table-mounted instruments. However, creating these precise
beam dimensions using a hand-held slit lamp is generally a
challenge. We note that a 1-mm circular beam aperture has
recently become available with the Kowa SL-17 hand-held
instrument, but this does not exactly mimic the dimensional
value of a 1 ·1 mm square beam. To best approximate the
optical section created using SUN’s settings with the most
widely used hand-held instruments, the SPOTS-modified
SUN system specifies use of a beam measuring 0.1 mm
Table 2. The Semiquantitative Preclinical
Ocular Toxicology Scoring–Modified
Standardization of Uveitis
Nomenclature Scoring System
The SPOTS system’s scheme for AC cells can be modified
to permit AC cell scoring with SUN’s original published
scheme.
1
This can be achieved using all currently
marketed hand-held slit lamp biomicroscopes. To do so,
the slit beam width is decreased to 0.1 mm and beam
height is reduced to 10 mm as determined by carefully
measuring the height of the beam using a mm ruler
imaged at the plane of focus. Assuming that the eye of the
animal being examined allows for imaging of the full
beam dimensions, this better approximates the optical
section volume produced by the 1 ·1 mm square beam of
the SUN system. In species with VCD <10 mm, the beam
height is adjusted accordingly.
Observation/lesion Score Description
Total number of AC cells viewed in volume (field) of the slit
beam
a,b
0£1 ells in field.
0.5 (trace) 1–5 cells in field.
1 6–15 cells in field.
2 16–25 cells in field.
3 26–50 cells in field.
4>50 cells in field.
a
Scoring of AC cell using the modified SUN scheme is performed
using the following slit lamp settings: 0.1 mm beam width, 10 mm
beam height, 30–45beam angle, and 10–16 ·magnification.
b
All AC cell scores should be accompanied by one of the
following notations of cell color: predominantly (*75%) white,
mixed color (*50% white, 50% brown and/or red), predominantly
(*75%) brown, or predominantly (*75%) red.
FIG. 8. (A, B) Photographic
exemplars demonstrating se-
vere (Score ‘‘3’’) perivascular
sheathing of the peripapillary
and nasal fundus (A) and
temporal fundus (B) in a NHP
using the SPOTS system.
THE SPOTS SCORING SYSTEMS 731
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
width and 10 mm height (as determined by carefully mea-
suring the height of the beam using a mm ruler imaged at the
plane of focus). Assuming that the eye of the animal being
examined allows for imaging of the full beam dimensions,
this better approximates the optical section volume pro-
duced by the 1 ·1 mm square beam of the SUN system.
SUN’s ordinal AC cell numeric scores are then used as
originally published (Table 2). It needs to be acknowledged
that the beam sample volume will differ between species
and that the beam area designated by the modified SUN
system cannot be achieved in rodents that have a VCD
<10 mm. As for the SPOTS AC cell scheme, cell color
should be noted with each assigned AC cell score.
Discussion
The SUN, M-S, H-M, and numerous other scoring systems
have been widely employed, but are associated with significant
limitations in the context of modern preclinical ocular drug
and device development. Individually, none integrate the full
complement of parameters that would be routinely evaluated
by slit lamp examination (see Table 1 in our accompanying
review of published scoring systems). Furthermore, each sys-
tem’s respective indications, methodologies, and species or
strain of interest confound application to the full range of
laboratory species and strains currently employed in vision
science and ocular drug development. The SPOTS systems
described in this study address many of these limitations.
Lack of consistency in slit lamp settings between exam-
iners introduces confounding variability when assessing the
anterior segment biomicroscopically, and designation of
such settings are lacking in the commonly used systems of
M-S and H-M. The SPOTS system specifies hand-held slit
lamp settings to ensure consistency in technique and optical
section volume evaluated. This is particularly relevant when
scoring AC cell density; the SPOTS system offers greater
granularity at the lower end of the scale, enhancing differ-
entiation between dose levels of a drug or test article in the
range that would likely be clinically tolerated.
Furthermore, AC cell scores using any system are influ-
enced by factors extrinsic to technique and instrument set-
tings. The most noteworthy of these factors is species-related
variation in anterior segment geometry, as reported in our
group’s recent geometric analysis of the effect of anatomy on
AC cell scoring using the SUN and SPOTS systems (refer-
enced in that analysis as the ‘‘OSOD systems’’) in a range of
laboratory species.
24
For example, in species with deeper
ACs, such as pigs, dogs, and cats, the optical section created
by slit beam illumination of the AC samples a comparatively
larger volume than in humans or NHPs. As a result, appli-
cation of the scoring schemes of both SUN and SPOTS-
Modified SUN yielded cell numbers in the pig, dog, and cat
that overestimated human values (as determined by calcu-
lating equivalent volume sampling between species), and cell
counts in mice and rats (with much smaller AC depths) that
underestimated human values. From these geometric models
the analysis presented a set of mathematically derived con-
version factors, normalized to the human that can be used to
facilitate comparison between species and translation be-
tween laboratory studies and human patients. It needs to be
noted that while this conversion accounts for numeric scoring
on a per-volume basis, it does not account for species vari-
ability in inflammatory response to a test article or device.
Developing a scoring system for vitreous cells presents a
unique challenge. Although vitreous cells are an important
marker of posterior segment inflammation, only the anterior
portion of the vitreous can be evaluated biomicroscopically.
Intrinsic to sampling of the anterior vitreous is a lack of
ability to control for exact depth of view as the slit beam is
swept across this region, preventing examination of a spe-
cific volume of vitreous. Also, cell dynamics in the vitreous
differ than those of the AC in that, once present, cells leave
the vitreous more slowly after cessation of dosing. Because
of this, vitreous cell number can continue to escalate with
sequential dosing while AC cell can peak and completely
resolve between doses.
We note that identification and semiquantification of cells
in the anterior vitreous does not necessarily reflect the pres-
ence (or absence) of the same population in other vitreous
regions that cannot be visualized; and a small population of
hyalocytes can be observed in normal eyes in some species
and can be quite marked as a normal finding in some rabbits.
Furthermore, vitreous volume, constituents, and rheology
differ considerably between species,
34,35
likely impacting the
translatability of a system’s vitreous cell scoring scheme
across species. Additionally, even if species differences in
intrinsic cellular response to a given test article could be
controlled for, there remains a knowledge gap regarding the
influence of differences in intrinsic vitreous chemistry and
rheology on cell dynamics in the vitreous. Semiquantification
and analysis of these variables is a complicated endeavor,
which likely contributes to the paucity of published anterior
vitreous scoring systems in the clinical and drug development
literature. In the absence of a validated scheme, however, we
have found consistent use of a vitreous cell scoring scheme
within the context of drug development programs to provide
useful comparator data between test articles and between-
dose levels of a given test article. It is imperative that the
beam width, height, angle, and magnification used for scoring
anterior vitreous cell be set and consistently employed
throughout a given study.
The anterior segment scoring systems of M-S and H-M,
although specifically developed for use in albino rabbits,
have been widely and successfully deployed in ocular drug/
device development programs. The SPOTS system seeks not
to replace the scoring schemes established by these investi-
gators, but instead to expand and refine their pioneering work
to improve applicability to multiple species, and to better
account for intraocular inflammation commonly encountered
in modern ocular drug development programs. The SPOTS
system’s criteria for corneal opacity and fluorescein staining
severity, for example, refine previously published schemes
which bear more relevance to ocular irritation associated
with industrial products as opposed to ocular pharmaceuti-
cals. Similarly, scoring of aqueous flare using schemes from
systems such as SUN, which are designed for naturally oc-
curring uveitis disease in human patients, does not ade-
quately capture the spectrum of findings encountered in
laboratory species used in drug development programs.
While our accompanying review article documents a wide
array of slit lamp-based scoring systems available in the
literature, there are remarkably few published scoring sys-
tems for the posterior segment. The genesis of many of the
preclinical scoring systems was motivated by determining
the safety of household, cosmetic, and industrial products,
and ocular irritancy scoring was the focus.
732 EATON ET AL.
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
There are limitations, principally related to ocular pig-
mentation in different laboratory species that the SPOTS
system currently does not address. For example, the presence
of perilimbal melanin in pigmented rabbits and NHPs may
hinder scoring of conjunctival hyperemia. Similarly, the
presence of melanin in the irides of pigmented laboratory
species will obscure view of smaller iridal vessels that would
otherwise be visible in albino species. However, as the pres-
ence of ocular pigment can have a considerable effect on the
pharmacologic properties of ocular drugs,
36–39
the Food and
Drug Administration currently requires preclinical evaluation
of an ocular drug or device in at least 1 pigmented species.
40
Further evolution of scoring systems will likely introduce
greater granularity with creation of species- and strain-
specific systems. Furthermore, as ocular imaging technolo-
gies such as in vivo confocal microscopy, optical coherence
tomography (OCT), and Scheimpflug imaging continue to be
refined, the objective and quantitative capabilities of these
instruments will likely lead to the development of instrument-
assisted scoring systems with the promise of even greater
reproducibility and translatability. Finally, the systems pre-
sented in this study, as well as all previously published sys-
tems, focus on consistency of findings within a species/strain
and do not directly address the predictive value of findings in
any 1 species/strain to human patients.
Summary
The SPOTS systems modify and expand upon previously
published ocular scoring systems, leveraging primarily the
systems of M-S and H-M as well as the SUN system. The
SPOTS systems evolved as an attempt to increase the appli-
cability and validity of these systems to a wider range of lab-
oratory animal species and strains, and incorporate ocular
findings commonly observed in preclinical studies involving
ocular drugs and devices. Standardization of slit lamp settings
and modification of settings to accommodate SUN scoring of
AC cell; introduction of novel scoring schemes for AC and
vitreous cell; refinement of previous scoring schemes for cor-
neal opacities, fluorescein staining, and aqueous flare; and
addition of ophthalmoscopic scoring criteria for the posterior
segment and IVT test articles collectively provide a system
with enhanced applicability in the context of modern drug
development.
Acknowledgments
The authors would like to thank Hugh Wabers and Mi-
chael Neider of OSOD, Monica Motta of the Murphy–
Russell–Thomasy Laboratory at the UC Davis School of
Veterinary Medicine, and the UC Davis Veterinary Oph-
thalmology Service for providing photographic images for
use in this publication; Chrisoula Toupadakis Skouritakis,
Director of the Media Laboratory at the UC Davis School of
Veterinary Medicine for assistance with figure design; and
T. Michael Nork, MD, MS of OSOD and the Department of
Ophthalmology & Visual Sciences at the University of
Wisconsin–Madison School of Medicine for his input and
expertise.
Proprietary Interest(s)
J.S.E.: Veterinary Ophthalmologist, OSOD; P.E.M.: Af-
filiate Veterinary Ophthalmologist, OSOD; E.B.: Affiliate
Veterinary Ophthalmologist, OSOD; S.M.T.: Affiliate Ve-
terinary Ophthalmologist, OSOD; C.J.M.: CEO and Affiliate
Veterinary Ophthalmologist, OSOD.
Author Disclosure Statement
No competing financial interests exist.
References
1. Jabs, D.A., Nussenblatt, R.B., and Rosenbaum, J.T. Stan-
dardization of uveitis nomenclature for reporting clinical
data. Results of the First International Workshop. Am. J.
Ophthalmol. 140:509–516, 2005.
2. Munger, R.J. Veterinary ophthalmology in laboratory ani-
mal studies. Vet. Ophthalmol. 5:167–175, 2002.
3. Munger, R.J., and Collins, M. Assessment of ocular toxicity
potential: basic theory and techniques. In: Weir, A.B., and
Collins M, eds. Assessing Ocular Toxicology in Laboratory
Animals. New York: Humana Press; 2013; p. 23–52.
4. Wilkie, D.A. The ophthalmic examination as it pertains to
general ocular toxicology: basic and advanced techniques
and species-associated findings. In: Gilger, B.C., ed. Ocular
Pharmacology and Toxicology. New York: Humana Press;
2014; p. 143–203.
5. McDonald, T.O., and Shadduck, J.A. Eye irritation. In:
Marzulli, F.N., and Maibach, H.I., eds. Advances in Mod-
ern Toxicology. Washington, D.C.: Hemisphere Publishing
Corp.; 1977; p. 139–191.
6. Hackett, R.B., and McDonald, T.O. Assessing ocular irrita-
tion. In: Marzulli, F.N., and Maibach, H.I., eds. Dermatotox-
icology. 5th ed. Washington, D.C.: Hemisphere Publishing
Corp.; 1996; p. 557–567.
7. Park, I.K., Chun, Y.S., Kim, K.G., et al. New clinical grading
scales and objective measurement for conjunctival injection.
Invest. Ophthalmol. Vis. Sci. 54:5249–5257, 2013.
8. Willingham, F.F., Cohen, K.L., Coggins, J.M., et al. Au-
tomatic quantitative measurement of ocular hyperemia.
Curr. Eye Res. 14:1101–1108, 1995.
9. Chun, Y.S., Yoon, W.B., Kim, K.G., et al. Objective As-
sessment of corneal staining using digital image analysis.
Invest. Ophthalmol. Vis. Sci. 55:7896–7903, 2014.
10. Zhao, W.J., Duan, F., Li, Z.T., et al. Evaluation of regional
bulbar redness using an image-based objective method. Int.
J. Ophthalmol. 7:71, 2014.
11. Li, Y., Lowder, C., Zhang, X., et al. Anterior chamber cell
grading by optical coherence tomography. Invest. Oph-
thalmol. Vis. Sci. 54:258–265, 2013.
12. Keane, P.A., Balaskas, K., Sim, D.A., et al. Automated anal-
ysis of vitreous inflammation using spectral-domain optical
coherence tomography. Trans. Vis. Sci. Tech. 4:4, 2015.
13. Dickson, D., Agarwal, A., Sadiq, M.A., et al. Assessment of
vitreous haze using ultra-wide field retinal imaging.
J. Ophthalmic. Inflamm. Infect. 6:35, 2016.
14. Waring, G.O., and Laibson, P.R. A systematic method of
drawing corneal pathologic conditions. Arch. Ophthalmol.
95:1540–1542, 1977.
15. Williams, D. Histological and immunohistochemical eval-
uation of canine chronic superficial keratitis. Res. Vet. Sci.
67:191–195, 1999.
16. Jakobiec, F.A., Stacy, R.C., Mendoza, P.R., et al. Hyper-
plastic corneal pannus: an immunohistochemical analysis
and review. Surv. Ophthalmol. 59:448–453, 2014.
17. Hogan, M.J., Kimura, S.J., and Thygeson, P. Signs and
symptoms of uveitis. I. Anterior uveitis. Am. J. Ophthalmol.
47:155–170, 1959.
THE SPOTS SCORING SYSTEMS 733
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
18. Kimura, S.J., Thygeson, P., and Hogan, M.J. Signs and
symptoms of uveitis. II. Classification of the posterior mani-
festations of uveitis. Am. J. Ophthalmol. 47:171–176, 1959.
19. Ghosn, C.R., Li, Y., Orilla, W.C., et al. Treatment of ex-
perimental anterior and intermediate uveitis by a dexa-
methasone intravitreal implant. Invest. Ophthalmol. Vis.
Sci. 52:2917–2923, 2011.
20. Eom, Y., Lee, D.Y., Kang, B.R., et al. Comparison of
aqueous levels of inflammatory mediators between toxic
anterior segment syndrome and endotoxin-induced uveitis
animal models. Invest. Ophthalmol. Vis. Sci. 55:6704–
6710, 2014.
21. Wang, J., Lu, H., Hu, X., et al. Nuclear factor translocation
and acute anterior uveitis. Mol. Vis. 17:170, 2011.
22. Short, B.G. Safety evaluation of ocular drug delivery for-
mulations: techniques and practical considerations. Toxicol.
Pathol. 36:49–62, 2008.
23. Weiner, A.L., and Gilger, B.C. Advancements in ocular
drug delivery. Vet. Ophthalmol. 13:395–406, 2010.
24. Thomasy, S.M., Eaton, J.S., Timberlake, M.J., et al. Spe-
cies differences in the geometry of the anterior segment
differentially affect anterior chamber cell scoring systems
in laboratory animals. J. Ocul. Pharmacol. Ther. 32:28–37,
2016.
25. Bito, L.Z. Species differences in the responses of the eye to
irritation and trauma: a hypothesis of divergence in ocular
defense mechanisms, and the choice of experimental ani-
mals for eye research. Exp. Eye Res. 39:807–829, 1984.
26. Murphy, C.J., and Rowland, H.C. The optics of compara-
tive ophthalmoscopy. Vis. Res. 27:599–607, 1987.
27. Friberg, T.R., and Yu, J.Y. Examination of the retina:
ophthalmoscopy and fundus biomicroscopy. In: Albert, D.,
Miller, J., Azar D et al., eds. Albert & Jakobiec’s Principles
& Practice of Ophthalmology. Philadelphia: Saunders; 2008.
28. Nussenblatt, R.B., Palestine, A.G., Chan, C.C., et al.
Standardization of vitreal inflammatory activity in inter-
mediate and posterior uveitis. Ophthalmology 92:467–471,
1985.
29. Buchen, S.Y., Calogero, D., Hilmantel, G., et al. Detecting
endotoxin contamination of ophthalmic viscosurgical de-
vices: intracameral versus intravitreal assays in rabbits.
Ophthalmology 119:e11–e18, 2012.
30. Ye, G.J., Budzynski, E., Sonnentag, P., et al. Safety and
biodistribution evaluation in cynomolgus macaques of
rAAV2tYF-PR1. 7-hCNGB3, a recombinant AAV vector
for treatment of achromatopsia. Hum. Gene Ther. Clin.
Dev. 27:37–48, 2016.
31. Leon, A. Diseases of the vitreous in the dog and cat. J.
Small Anim. Pract. 29:448–461, 1988.
32. Agarwal, M., Malathi, J., and Biswas, J. Frosted branch
angiitis in a patient with typhoid fever. Ocul. Immunol.
Inflamm. 1–3, 2016.
33. Wood, E.H., and Wong, R.W. Bilateral frosted branch an-
giitis as the presenting sign of antiphospholipid antibody
syndrome. J. Ophthalmic. Inflamm. Infect. 6:20, 2016.
34. Ve
´zina, M. Comparative ocular anatomy in commonly used
laboratory animals. In: Weir, A.B., and Collins M, eds.
Assessing Ocular Toxicology in Laboratory Animals. New
York: Humana Press; 2013; p. 1–21.
35. Pfeffer, B.A., Bernstein, S.A., and Bartels, S.P. Preservation
of structure and immunoreactivity at the vitreoretinal inter-
face of the rabbit eye. Graefe’s Arch. Clin. Exp. Ophthalmol.
247:193–205, 2009.
36. Sasaki, H., Yamamura, K., Nishida, K., et al. Delivery of
drugs to the eye by topical application. Prog. Retin. Eye
Res. 15:583–620, 1996.
37. Salminen, L., Urtti, A., and Perivita, L. Effect of ocular
pigmentation on pilocarpine pharmacology in the rabbit
eye. I. Drag distribution and metabolism. Int. J. Pharm. 18:
17–24, 1984.
38. Urtti, A., Salminen, L., Kujari, H., et al. Effect of ocular
pigmentation on pilocarpine pharmacology in the rabbit
eye. II. Drug response. Int. J. Pharm. 19:53–61, 1984.
39. Katz, I.M., and Berger, E.T. Effects of iris pigmentation on
response of ocular pressure to timolol. Surv. Ophthalmol.
23:395–398, 1979.
40. Mirowski, P., and Van Horn, R. The contract research or-
ganization and the commercialization of scientific research.
Soc. Stud. Sci. 35:503–548, 2005.
41. Krzystolik, M.G., Afshari, M.A., Adamis, A.P., et al. Pre-
vention of experimental choroidal neovascularization with
intravitreal anti-vascular endothelial growth factor antibody
fragment. Arch. Ophthalmol. 120:338–346, 2002.
42. Nork, T.M., Dubielzig, R.R., Christian, B.J., et al. Pre-
vention of experimental choroidal neovascularization and
resolution of active lesions by VEGF trap in nonhuman
primates. Arch. Ophthalmol. 129:1042–1052, 2011.
43. Ryan, S.J. Subretinal neovascularization: natural history of
an experimental model. Arch. Ophthalmol. 100:1804–1809,
1982.
44. Ohkuma, H., and Ryan, S.J. Experimental subretinal neo-
vascularization in the monkey: permeability of new vessels.
Arch. Ophthalmol. 101:1102–1110, 1983.
Received: September 11, 2017
Accepted: October 17, 2017
Address correspondence to:
Dr. Joshua Seth Eaton
Ocular Services On Demand (OSOD), LLC
6527 Normandy Lane, Suite 100
Madison, WI 53719
E-mail: seaton@ocularservices.com
Dr. Christopher J. Murphy
Department of Surgical & Radiological Sciences
School of Veterinary Medicine
University of California—Davis
1 Shields Avenue
Davis, CA 95616
E-mail: cjmurphy@ucdavis.edu
734 EATON ET AL.
Downloaded by Uc Davis Libraries University of California Davis from online.liebertpub.com at 12/15/17. For personal use only.
... Our results are subjective due to the retrospective nature of the study and lack of standardized scoring for this ocular disease in dogs, and may have been under-or overestimated in some cases. Future work should focus on a simplified grading scheme that is reproducible and clinically useful, as exemplified by scoring systems reported by Eaton et al. and Sebbag et al. [36,37], ideally correlating with the different therapeutical protocols and this grading scheme could be used by multiple evaluators for additional validation. ...
Article
Full-text available
Background In humans, allergic conjunctivitis is a well described disease. In contrast, allergic conjunctivitis has not received much attention from the veterinary community so far. Canine allergic conjunctivitis (cAC) is one of the possible manifestations associated with canine atopic dermatitis (cAD), being often underdiagnosed and undertreated. Our aim is to contribute to disease characterization and clinical stagingfor cAC severity. Results A retrospective observational study including 122 dogs that underwent a complete ophthalmological and dermatological examinations and diagnosed with allergic conjunctivitis was conducted. A total of six ophthalmic clinical signs were considered for disease characterization and clinical staging: conjunctival hyperemia, chemosis, ocular pruritus, epiphora, seromucoid to mucopurulent discharge and keratitis, classified from 0 (absent) to 3 (severe). Scores comprised between 1–5 were considered mild, 6–10 moderate and 11–18 severe. The majority of dogs (64%) presented with moderate cAC followed by 24% of mild stages and only 12% of severe presentations. The severity of allergic conjunctivitis was not correlated to sex or age at the time of diagnosis and all presented with a bilateral form of the disease. Chemosis (84%), hyperemia (83%) and ocular pruritus (79%) was observed in 55% of the cases. Seromucoid to mucopurulent discharge (62%) and epiphora (69%) were less frequent, with keratitis being the least encountered clinical sign (15%). The degree of keratitis showed a positive correlation with both severity and chronicity of cAC (rho = 0.21–0.29, p ≤ 0.02)). Severity of cAD and cAD were not significantly correlated (p-value = 0.4). Discussion and conclusion The triad hyperemia, chemosis and ocular pruritus, already known in human medicine to be a reliable way of diagnosing human allergic conjunctivitis, also proved to be important in cAC Mild forms of the disease may pass unnoticed, ocular pruritus being hard to assess in canine patients.The proposed standardized diagnostic approach and novel grading scheme for cAC may be of value for both veterinary ophthalmologists and dermatologists, as well as general practitioners.
... A standardized scoring system, accounting for species-specific differences in anterior segment anatomy, is used. 85,86 IOP, measured by tonometry, provides a functional assessment of both the ciliary body epithelium's ability to produce AH and the structures of the aqueous outflow pathways to remove it. Results should continuously be reviewed to justify redosing versus therapeutic intervention/euthanasia for animal welfare reasons. ...
Article
The eye, which is under constant exposure to environmental pathogens, has evolved various anatomical and immunological barriers critical to the protection of tissues lacking regenerative capacity, and the maintenance of a clear optic pathway essential to vision. By bypassing the ocular barriers, intravitreal (IVT) injection has become the mainstay for the delivery of drugs to treat conditions that affect the back of the eye. Both small molecules and biotherapeutics have been successfully administered intravitreally, and several drugs have been approved for the treatment of (wet) age-related macular degeneration (AMD) and diabetic macular edema (DME). However, IVT injection is an invasive procedure, which requires sufficient technical expertise from the healthcare professional administering the drug. Potential side effects include bleeding, retinal tear, cataracts, infection, uveitis, loss of vision, increased ocular pressure. Pharmaceutical companies often differ in their drug development plan, including drug administration techniques, collection of ocular tissues and fluids, ophthalmology monitoring and overall conduct of nonclinical and clinical studies. The present effort, under the aegis of the Innovation & Quality Ophthalmic Working Group, aims at understanding these differences, identifying pros and cons of the various approaches, determining the gaps in knowledge, and suggesting feasible good practices for non-clinical and early clinical IVT drug development.
... Ophthalmic observations and scoring of findings are generally performed as a mixture of grades of inflammation according to established and acceptable scoring systems (i.e. modified Standardization of Uveitis Nomenclature [SUN] scoring system) [144], or McDonald-Shadduck and Hackett-McDonald scoring systems [145], with photos or detailed drawings describing any ocular reactions within the globe of the eye. ...
Article
Introduction: Ophthalmic diseases of the retina are a significant cause of vision loss globally. Despite much progress, there remains an unmet need for durable, long-acting treatment options. While biologic therapies show great promise, they present many challenges, including complexities in biochemical properties, mechanism of action, manufacturing considerations, preclinical evaluation, and delivery mechanism; these are confounded by the unique anatomy and physiology of the eye itself. Areas covered: This review describes the current development status of intravitreally administered drugs for the treatment of ophthalmic disease, outlines the range of approaches that can be considered for sustained drug delivery to the eye, and discusses key preclinical considerations for the evaluation of ocular biologics. Expert opinion: The required frequency of dosing in the eye results in a great burden on both patients and the health care system, with direct intraocular administration remaining the most reliable and predictable route. Sustained and controlled ophthalmic drug delivery systems will go a long way in reducing this burden. Sustained delivery can directly dose target tissues, improving bioavailability and reducing off-target systemic effects. Maintaining stability and activity of compounds can prevent aggregation and enable extended duration of release, while sustaining dosage and preventing residual polymer after drug depletion.
... 110,111 PFS was described in veterinary medicine by Saito and Kotani who showed that corneal epitheliopathy was observed in 97% of dogs with epiphora and 55% of dogs without epiphora. 112 Since then, the use of PFS was fine-tuned with the development of various grading schemes: (i) In the comprehensive SPOTS system reported by Eaton et al., 113 the presence of corneal fluorescein uptake was defined as grade 0 (absence of punctate staining), grade 1 (slight punctate staining), grade 2 (apparent multifocal to coalescent punctate staining), grade 3 (epithelial loss without stromal loss), and grade 4 (epithelial and stromal loss) 113 ; (ii) Saito et al. described PFS in regard to the stain intensity (grade 0 indicating "absence of punctate staining" to grade 3 indicating "severe large dots and/or linear punctate staining") and the area of the cornea affected by punctate staining: grade 0 (absence of punctate staining), grade 1 (less than 50% area of staining), and grade 2 (more than 50% area of staining) 73 ; (iii) Sebbag et al. used a slight modification of the aforementioned grading systems, describing PFS in Shih Tzus as grade 1 (discrete stain, <1/3 of cornea), grade 2 (moderate stain, 1/3 to 2/3 of cornea), and grade 3 (diffuse stain, >2/3 of cornea). 23 Saito et al. also used PFS to describe changes in the upper palpebral conjunctiva of dogs, 73 identifying staining patterns that resemble a condition described in human patients (lid wiper epitheliopathy). ...
Article
Dry eye disease (DED) is a complex multifactorial condition caused by loss of ocular surface homeostasis from quantitative and/or qualitative tear film deficiency. Schirmer tear test (STT) is often the only diagnostic test used to assess for DED in veterinary practice. STT is invaluable in the diagnosis and monitoring of quantitative tear film deficiency (i.e., keratoconjunctivitis sicca); however, it is not sufficient to optimize therapy and fully recognize other contributing factors for the disturbance in ocular surface homeostasis. The present work reviews diagnostic tests for assessing aqueous tear production in veterinary medicine, as well as the quality of tears, corneal epithelial barrier integrity, and the lacrimal functional unit.
... Conjunctival hyperemia scores were assigned to each eye at initial exam and prior to each treatment. Eyes were scored (0-4 +) independently by the investigators (KW, BM) according to the semiquantitative preclinical ocular toxicology score (SPOTS) system [37]. Images of patient eyes were obtained with a digital camera (Nikon D160 DSLR, micro Nikkor 105 mm lens, Nikon Inc., Melville, NY). ...
Article
Full-text available
Ocular surface squamous neoplasia (OSSN) is the major cause of corneal cancer in man and horses worldwide, and the prevalence of OSSN is increasing due to greater UVB exposure globally. Currently, there are no approved treatments for OSSN in either species, and most patients are managed with surgical excision or off-label treatment with locally injected interferon alpha, or topically applied cytotoxic drugs such as mitomycin C. A more broadly effective and readily applied immunotherapy could exert a significant impact on management of OSSN worldwide. We therefore evaluated the effectiveness of a liposomal TLR complex (LTC) immunotherapy, which previously demonstrated strong antiviral activity in multiple animal models following mucosal application, for ocular antitumor activity in a horse spontaneous OSSN model. In vitro studies demonstrated strong activation of interferon responses in horse leukocytes by LTC and suppression of OSSN cell growth and migration. In a trial of 8 horses (9 eyes), treatment with topical or perilesional LTC resulted in an overall tumor response rate of 67%, including durable regression of large OSSN tumors. Repeated treatment with LTC ocular immunotherapy was also very well tolerated clinically. We conclude therefore that ocular immunotherapy with LTC warrants further investigation as a novel approach to management of OSSN in humans.
Article
Ocular tissue is highly sensitive to chemical exposures. Chloropicrin (CP), a choking agent employed during World War I and currently a popular pesticide and fumigating agent, is a potential chemical threat agent. Accidental, occupational, or intentional exposure to CP results in severe ocular injury, especially to the cornea; however, studies on ocular injury progression and underlying mechanisms in a relevant in vivo animal model are lacking. This has impaired the development of effective therapies to treat the acute and long-term ocular toxicity of CP. To study the in vivo clinical and biological effects of CP ocular exposure, we tested different CP exposure doses and durations in mice. These exposures will aid in the study of acute ocular injury and its progression as well as identify a moderate dose to develop a relevant rodent ocular injury model with CP. The left eyes of male BALB/c mice were exposed to CP (20% CP for 0.5 or 1 min or 10% CP for 1 min) using a vapor cap, with the right eyes serving as controls. Injury progression was evaluated for 25 days post-exposure. CP-exposure caused a significant corneal ulceration and eyelid swelling which resolved by day 14 post exposure. In addition, CP-exposure caused significant corneal opacity and neovascularization. Development of hydrops (severe corneal edema with corneal bullae) and hyphema (blood accumulation in the anterior chamber) was observed as advanced CP effects. Mice were euthanized at day 25 post-CP-exposure, and the eyes were harvested to further study the corneal injury. Histopathological analyses showed a significant CP-induced decrease in corneal epithelial thickness and increased stromal thickness with more pronounced damage, including stromal fibrosis, edema, neovascularization, trapped epithelial cells, anterior and posterior synechiae, and infiltration of inflammatory cells. Loss of the corneal endothelial cells and Descemet's membrane could be associated with the CP-induced corneal edema and hydrops which could lead to long term term pathological conditions. Although exposure to 20% CP for 1 min caused more eyelid swelling, ulceration, and hyphema, similar effects were observed with all CP exposures. These novel findings following CP ocular exposure in a mouse model outline the corneal histopathologic changes that associate with the continuing ocular clinical effects. The data are useful in designing further studies to identify and correlate the clinical and biological markers of CP ocular injury progression with acute and long-term toxic effects on cornea and other ocular tissues. We take a crucial step towards CP ocular injury model development and in pathophysiological studies to identify molecular targets for therapeutic interventions.
Preprint
Full-text available
We propose FedScore, a privacy-preserving federated learning framework for scoring system generation across multiple sites to facilitate cross-institutional collaborations. The FedScore framework includes five modules: federated variable ranking, federated variable transformation, federated score derivation, federated model selection and federated model evaluation. To illustrate usage and assess FedScore's performance, we built a hypothetical global scoring system for mortality prediction within 30 days after a visit to an emergency department using 10 simulated sites divided from a tertiary hospital in Singapore. We employed a pre-existing score generator to construct 10 local scoring systems independently at each site and we also developed a scoring system using centralized data for comparison. We compared the acquired FedScore model's performance with that of other scoring models using the receiver operating characteristic (ROC) analysis. The FedScore model achieved an average area under the curve (AUC) value of 0.763 across all sites, with a standard deviation (SD) of 0.020. We also calculated the average AUC values and SDs for each local model, and the FedScore model showed promising accuracy and stability with a high average AUC value which was closest to the one of the pooled model and SD which was lower than that of most local models. This study demonstrates that FedScore is a privacy-preserving scoring system generator with potentially good generalizability.
Article
Corneal wound healing is integral for resolution of corneal disease or for post-operative healing. However, corneal scarring that may occur secondary to this process can significantly impair vision. Tissue transglutaminase 2 (TGM2) inhibition has shown promising antifibrotic effects and thus holds promise to prevent or treat corneal scarring. The commercially available ocular solution for treatment of ocular manifestations of Cystinosis, Cystaran®, contains the TGM2 inhibitor cysteamine hydrochloride (CH). The purpose of this study is to assess the safety of CH on corneal epithelial and stromal wounds, its effects on corneal wound healing, and its efficacy against corneal scarring following wounding. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were first used to quantify and localize TGM2 expression in the cornea. Subsequently, (i) the in vitro effects of CH at 0.163, 1.63, and 16.3 mM on corneal epithelial cell migration was assessed with an epithelial cell migration assay, and (ii) the in vivo effects of application of 1.63 mM CH on epithelial and stromal wounds was assessed in a rabbit model with ophthalmic examinations, inflammation scoring, color and fluorescein imaging, optical coherence tomography (OCT), and confocal biomicroscopy. Post-mortem assessment of corneal tissue post-stromal wounding included biomechanical characterization (atomic force microscopy (AFM)), histology (H&E staining), and determining incidence of myofibroblasts (immunostaining against α-SMA) in wounded corneal tissue. TGM2 expression was highest in corneal epithelial cells. Application of the TGM2 inhibitor CH did not affect in vitro epithelial cell migration at the two lower concentrations tested. At 16.3 mM, decreased cell migration was observed. In vivo application of CH at 57 mM was well tolerated and did not adversely affect wound healing. No difference in corneal scarring was found between CH treated and vehicle control eyes. This study shows that the TGM2 inhibitor CH, at the FDA-approved dose, is well tolerated in a rabbit model of corneal wound healing and does not adversely affect epithelial or stromal wound healing. This supports the safe use of this medication in Cystinosis patients with open corneal wounds. CH did not have an effect on corneal scarring in this study, suggesting that Cystaran® administration to patients with corneal wounds is unlikely to decrease corneal fibrosis.
Article
Regenerative medicine requires both tissue restoration and ease of compliance for clinical application. Considering this, sticky tissue sealants have been shown to have great potentials over surgical suturing and wound treatment. However, tissue sealants currently used pose challenges such as uncontrollable adhesion formation, mechanical mismatch, and lack of tissue restoration. A new sticky sealant based on gelatinized cornea-derived extracellular matrix (GelCodE) with a visible light-activating system is firstly being introduced in this study. De novo tissue regeneration relies on the matrisome in charge of tissue-organization and development within GelCodE while visible light-based photopolymerization with ruthenium/sodium persulfate rapidly induces covalent bonds with the adjacent tissues. The ease of not only in vivo application, biocompatibility, and biointegration, but also exceptional de novo tissue formation is demonstrated in this study. Interestingly, newly regenerated tissues were shown to have normal tissue-like matrices with little scar formation. Hence, this work presents a promising strategy to meet clinical demands for scar-free tissue recovery with superior ease of clinical application.
Article
Scleras are mainly used for the treatment of glaucoma, eyelid damage, and scleral ulcers. Given that the sclera and cornea collectively constitute the complete external structure of the eyeball and both have the same tissue and cell origin, we attempted to identify scleral materials to treat lamellar and penetrating corneal injuries. Based on research in our center, antigenic components in decellularized porcine sclera (DPS) were removed using a simplified decellularization method, leaving the collagen structure and active components undamaged. DPS preserved the mechanical properties and did not significantly inhibit the proliferation and replication of human corneal epithelial cells. In vivo, the graft epithelium healed well after lamellar and penetrating scleral grafting, and the graft thickness did not change evidently. DPS can resist suture traction during scleral transplantation and maintain anterior chamber stability until day 28 post-operatively, especially in penetrating repairs. No obvious immune rejection of lamellar or penetrating scleral grafts was found 28 days after DPS transplantation. This study shows that DPS could be used as an alternative material for the emergency repair of corneal perforations and lamellar injuries, representing another application of sclera.
Article
Full-text available
Background Conventional fundus imaging has been used to assess vitreous haze (VH) in patients with uveitis. Ultra-wide field (UWF) retinal imaging that uses scanning laser technology has not been evaluated for the detection of VH. This pilot study evaluates the ability of UWF imaging in detecting VH.Patients with intermediate, posterior, or panuveitis were examined to assess the level of VH using slit-lamp biomicroscopy. Colored fundus images were acquired using a Carl Zeiss FF450 camera. The same photographer obtained fundus images of the same eyes during the same visit by Optos UWF P200Tx retinal camera. Two graders independently analyzed UWF fundus images for presence or absence of VH, without quantifying the degree of VH using any scale. The images were analyzed using the composite red plus red-free wavelengths utilized by the Optos UWF camera and by using each wavelength exclusively. These findings were compared to clinical detection of VH and detection of VH using conventional fundus photography. ResultsNinety-two eyes were included in the study. For composite UWF images, sensitivity was 0.27, specificity was 0.88, PPV was 0.31, NPV was 0.86, positive LR was 2.25, and negative LR was 0.83. For the conventional Zeiss images, sensitivity was 0.5, specificity was 0.84, PPV was 0.33, NPV was 0.91, positive LR was 3.13, and negative LR was 0.6.Agreement between the composite UWF and Zeiss techniques was substantial with k = 0.64. Inter-observer agreement for composite UWF images was also substantial with k = 0.65. Inter-observer agreement for Zeiss images was moderate with k = 0.471. Intra-observer agreement for both imaging modalities was substantial with a composite UWF k = 0.76 and Zeiss k = 0.7. ConclusionsUWF fundus imaging using scanning laser technique may be used to assess VH and employed in the management of intermediate, posterior, and panuveitis.
Article
Full-text available
Background: "Frosted branch retinal angiitis" is an encompassing term for a rare, typically bilateral diffuse retinal periphlebitis that may occur in a number of varying conditions. To our knowledge, we report the first case of frosted branch angiitis as the presenting sign of antiphospholipid antibody syndrome in a 28-year-old woman. Findings: This study is a retrospective case report and literature review. Serial fundus photos, fluorescein angiogram, and ocular coherence tomography taken were before and after treatment, showing resolution of diffuse retinal perivascular sheathing and macular edema along with marked improvement in visual acuity 4 months after the treatment with corticosteroids. Conclusions: Frosted branch angiitis can be seen in association with antiphospholipid antibody syndrome. Prompt recognition and treatment with corticosteroids may result in good visual prognosis, and long-term immunosuppression and additional anticoagulation may be beneficial to prevent recurrence.
Article
Full-text available
Purpose: To determine the impact of anterior segment geometry on ocular scoring systems quantifying anterior chamber (AC) cells in humans and 7 common laboratory species. Methods: Using normative anterior segment dimensions and novel geometric formulae, ocular section volumes measured by 3 scoring systems; Standardization of Uveitis Nomenclature (SUN), Ocular Services On Demand (OSOD), and OSOD-modified SUN were calculated for each species, respectively. Calculated volumes were applied to each system's AC cell scoring scheme to determine comparative cell density (cells/mm(3)). Cell density values for all laboratory species were normalized to human values and conversion factors derived to create modified scoring schemes, facilitating interspecies comparison with each system, respectively. Results: Differences in anterior segment geometry resulted in marked differences in optical section volume measured. Volumes were smaller in rodents than dogs and cats, but represented a comparatively larger percentage of AC volume. AC cell density (cells/mm(3)) varied between species. Using the SUN and OSOD-modified SUN systems, values in the pig, dog, and cat underestimated human values; values in rodents overestimated human values. Modified normalized scoring systems presented here account for species-related anterior segment geometry and facilitate both intra- and interspecies analysis, as well as translational comparison. Conclusions: Employment of modified AC cell scoring systems that account for species-specific differences in anterior segment anatomy would harmonize findings across species and may be more predictive for determining ocular toxicological consequences in ocular drug and device development programs.
Article
Full-text available
Purpose: To develop an automated method for quantifying vitreous signal intensity on optical coherence tomography (OCT), with particular application for use in the assessment of vitreous inflammation. Methods: This retrospective, observational case-control series comprised 30 patients (30 eyes), with vitreous haze secondary to intermediate, posterior, or panuveitis; 12 patients (12 eyes) with uveitis without evidence of vitreous haze; and 18 patients (18 eyes) without intraocular inflammation or vitreoretinal disease. The presence and severity of vitreous haze was classified according to the National Eye Institute system; other inflammatory indices and clinical parameters were also documented. Spectral-domain OCT images were analyzed using custom VITreous ANalysis software (termed 'VITAN'), which is fully automated and avoids the need for manual segmentation. Results: VITAN performed accurate segmentation in all scans. Automated measurements of the vitreous:retinal pigment epithelium (RPE) signal ratio showed a moderate correlation with clinical vitreous haze scores (r = 0.585, P < 0.001), comparable to that reported using manual segmentation in our previous study (r = 0.566, P = 0.0001). The novel parameter of vitreous:RPE textural ratio showed a marginally stronger correlation (r = 0.604, P < 0.001) with clinical vitreous haze scores than the Vitreous:RPE signal ratio. Conclusions: The custom OCT image analysis software (VITAN) allows rapid and automated measurement of vitreous parameters, that is comparable to our previously reported vitreous:RPE index, and correlates with clinically measured disease activity. Such OCT-based indices may provide the much needed objective markers of vitreous activity, which may be used in both clinical assessment, and as outcome measures in clinical trials for intermediate, posterior, and panuveitis. Translational relevance: We describe a rapid automated method for quantifying vitreous signal intensity on optical coherence tomography (OCT) and show that this correlates with clinical assessment of vitreous inflammation. Such OCT-based indices may provide the much needed objective markers of vitreous activity, which may be used both in routine clinical assessment, and as outcome measures in clinical trials for intermediate, posterior, and panuveitis.
Article
Frosted branch angiitis (FBA), a rare form of retinal vasculitis presenting as bilateral perivascular sheathing, resembling the appearance of frosted tree branches in winter, was first reported by Ito et al.(1) in 1976, in a young immunocompetent boy. FBA predominantly affects healthy young patients, the youngest reported in an 11-month-old infant(2) and oldest in a 42-year-old patient.(3) Classical symptoms include sudden onset of blurred vision with floaters and photopsiae. Fundus examination shows widespread perivascular translucent sheathing affecting both arterioles and venules, more commonly latter. Fluorescein angiography shows late staining of vessels with no obstruction of blood flow. Electroretinogram shows reduced amplitude and visual fields show generalized constriction. Medline search did not show any case of frosted branch angiitis in a patient with typhoid fever.
Article
Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated viral (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. Three groups of animals (n = 2 males and 2 females per group) received a subretinal injection in one eye of 300 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 10¹¹ or 4 × 10¹² vector genomes/ml) and were evaluated over a 3-month period before being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on study day 5, based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.
Article
To begin a process of standardizing the methods for reporting clinical data in the field of uveitis. Consensus workshop. Members of an international working group were surveyed about diagnostic terminology, inflammation grading schema, and outcome measures, and the results used to develop a series of proposals to better standardize the use of these entities. Small groups employed nominal group techniques to achieve consensus on several of these issues. The group affirmed that an anatomic classification of uveitis should be used as a framework for subsequent work on diagnostic criteria for specific uveitic syndromes, and that the classification of uveitis entities should be on the basis of the location of the inflammation and not on the presence of structural complications. Issues regarding the use of the terms "intermediate uveitis," "pars planitis," "panuveitis," and descriptors of the onset and course of the uveitis were addressed. The following were adopted: standardized grading schema for anterior chamber cells, anterior chamber flare, and for vitreous haze; standardized methods of recording structural complications of uveitis; standardized definitions of outcomes, including "inactive" inflammation, "improvement'; and "worsening" of the inflammation, and "corticosteroid sparing," and standardized guidelines for reporting visual acuity outcomes. A process of standardizing the approach to reporting clinical data in uveitis research has begun, and several terms have been standardized.
Article
Ocular toxicology pertains to toxicologic effects of drugs administered topically, intraocularly, or systemically. It should also include evaluation of adverse effects of ophthalmic devices such as contact lenses, intraocular lenses, and glaucoma implants. The ophthalmic examination is able to provide detailed in-life information and is used in combination with clinical observations, clinical pathology, and histopathology to assess potential toxicologic effects. The ophthalmologist must be familiar with the wide range of species used in the field of toxicology, be familiar with the anatomic variations associated with these species, be able to determine what is an inherited or a breed-related finding from a study-related effect, be competent with the required ophthalmic equipment, and be capable of examining this wide range of animals.