Design and Conduct of Preclinical and Clinical Trial in Opthalmology
Design and Conduct of
Preclinical and Clinical
Trials in Opthalmology
What is ophthalmology?
Logos=word, thought, discourse
The science of eyes is opthalmology
The branch of medicine concerned with
Procedures involved in clinical trials
Eye diseases are among the most
devastating disorders leading to retinal
ganglion cell (RGC) degeneration, visual field
loss and, potentially, blindness. The
pathophysiologic mechanisms underlying
optic neuropathies are not fully understood,
although research in this area is substantial.
Animal models are required in order to
investigate the mechanism of each disease
in detail, to improve our knowledge of how
and why optic nerve axons die, and to test
new treatment modalities. Developing an
animal model for a disease, however, is
usually complicated and challenging, and
most experimental models are remote from
the human disease.
There are several animal species that can be
developed as suitable models for human
disease. The advantage of using a monkey is
its close phylogeny and high homology with
Monkeys have retinal and optic nerve
anatomy that is nearly identical to human
The validity of the experimental model,
however, depends upon the degree to which
it emulates the human condition, and a
primate model is considered as having the
closest compatibility for conducting research
with the goal of understanding human
Indeed, testing new treatment modalities in a
monkey model is usually the last step before
embarking upon clinical trials in humans.
Unfortunately, monkeys are very expensive,
their availability is limited, and they are
difficult to handle. Experiments in monkeys
require highly experienced teams and special
housing facilities, making them beyond the
reach of many research laboratories.
Thus, rats and mice are commonly used to
investigate optic nerve diseases. There is
great conservation between rats, mice,
and human genomes, which allows them
to be widely used for research on human
optic nerve disease.
While rats are commonly used to study optic
nerve injuries, the availability of a mouse
model confers unique advantages. Genetic
studies in mice are proving to be a potent
means for learning about genes and
pathologic mechanisms that cause disease.
In addition, the genome of the mouse can be
altered by adding transgenes or altering
Laboratory mice live for approximately 2
years and often develop diseases that
take decades to develop in humans.
Models of optic nerve disease in the
mouse can enable us to study the
mechanism of RGC degeneration and
potential new therapies using genetic
manipulation in various transgenic and
knockout mice that are not available in
Experimental glaucoma in monkeys
While the most useful model for experimental
glaucoma in monkeys is produced by argon
laser photocoagulation treatment to the
trabecular meshwork. The model was first
suggested by Gaasterland and Kupfer and
later refined by Quigley and Hohman. In
order to induce elevated IOP, monkeys are
sedated and the laser beam is focused as
precisely as possible on the centre of the
The recommended laser setting is a 50 m
spot diameter, 1–1.5 W, and a 0.5-s
duration. In this model, the treated eyes
develop sustained, moderately elevated IOP
with decreased outflow facility and optic
nerve cupping such as that in human
IOP can be measured under sedation and
topical anaesthesia using the Goldman
applanation tonometer. Optic nerve
damage can be evaluated by stereoscopic
ophthalmoscopy, fundus photographs,
nerve fibre layer photographs, axonal
counting by image analysis system, and
A second model for producing chronic
experimental glaucoma in primates was
developed by Quigley and Addicks. In this
model, extended IOP elevation was produced
by injection of autologous, fixed red blood
cells (RBC), and ghost red cells into the
anterior chamber. Filling of the anterior
chamber with blood, however, leads to the
disadvantage of poor visibility on the optic
nerve head and retina.
Experimental glaucoma in rats
Sustained increased IOP is
achieved by treating the
outflow channels of the
rat eye through the
peripheral cornea with a
diode laser at 532 nm
with a laser setting of
0.7 s, 0.4 W, and a spot
size of 50 m.
The laser beam is delivered from a slit–
lamp system without additional lenses and
with the animal under general
The rat eye is rotated manually to allow
the laser beam to be directed in a sharp
angle to the trabecular meshwork (TM).
Treatment takes approximately 3 min per eye
by an experienced researcher, therefore
many rats can be treated in 1 day.
The laser treatment is given unilaterally and
is repeated after a week if the IOP difference
between the treated eye and the fellow eye is
less than 6 mmHg. IOP is measured with the
Tonopen XL in both eyes before and
immediately after laser treatment, every 3–4
days in the first 2 weeks, and weekly
thereafter. In all, 10 measurements are
obtained for each eye from which the mean
There are three additional currently available
models for experimental glaucoma in rats, but all
of them are more time consuming and technically
more difficult to reproduce than the laser
Morrison and co-workers increased rat IOP by
hyperosmotic saline microinjection into the
episcleral veins of Brown–Norway rats in order to
increase the outflow resistance.
This was sufficient for about 50% of rats to
develop elevated IOP, even though most
animals needed repeated injections. The
advantage of using Brown–Norway rats is
that IOP can be measured by a Tonopen –XL
on awake animals. General anaesthetics
were shown to have resulted in a rapid and
substantial decrease in IOP in all eyes, and
so measurements of IOP in awake animals
provide the most accurate documentation of
IOP history for rat glaucoma model studies.
Another rodent model for chronic glaucoma
was developed by Ueda et al. They injected
India ink into the anterior chamber 1 week
prior to laser photocoagulation treatment.
Increased IOP was achieved in all rats within
4 weeks and optic nerve damage developed
With recent advances in genetic
manipulation, the development of
experimental glaucoma in mice became a
matter of high priority. One of the main
obstacles was measuring IOP in the mouse
eye. Several devices were recently
developed to measure IOP in a noninvasive
way, all claiming to have the ability to
measure IOP accurately in mice.
Some use the Tonopen without the ocufilm
cover, while others use a modified
Goldmann tonometer, both with reliable
Another model that has become well
established over the past few years was
developed by John et al. It consists of the
mouse strain DBA/2J that develops
glaucoma subsequent to anterior chamber
changes. The glaucoma in the DBA/2J mice
shares similarities with pigmentary
dispersion glaucoma in humans.
These mice develop pigment dispersion, iris
transillumination, iris atrophy, anterior
synechia, and elevated IOP. The prevalence
and severity increase with age, followed by
RGC death, and optic nerve degeneration.
Two chromosomal regions, one on
chromosome 6 and one on chromosome 4,
contribute to this glaucoma model.
Recently, John and his group generated and
studied mouse models with mutations in
genes that are responsible for anterior
segment dysgenesis anomalies in humans.
Developing these mouse models supports
the search for mutations in specific human
candidate genes. Although these models are
specific for unique types of glaucoma, they
can be used to test the mechanism of RGC
death in glaucoma as well as to evaluate
new treatment modalities.
Optic nerve transection
In rats, optic nerve transection is usually
performed using an intraorbital
approach. Using a binocular operating
microscope, the superior conjunctiva is
incised, the muscles and connective tissue
are separated, and the intraorbital optic
nerve is exposed.
A blade knife is used to transect the optic
nerve behind the globe, taking care not to
interfere with the blood supply and sparing
the meningeal sheaths. At the end of the
procedure, the retinas should be examined
ophthalmoscopically to assure blood vessel
RGCs degenerate rapidly after optic nerve
transection. One–half of them will
degenerate 4 days after optic nerve
axotomy, and it rises to about 70% in 1
week and to more than 95% in 2 weeks.
In monkeys, the optic nerve is transected
by a lateral approach through the skin and
the bone of the orbit approximately 6 mm
posterior to the globe. The rate of RGC
degeneration in this model is usually
slower, with about a 70% loss at 1 month
and a 90% loss at 2 months.
Ischaemic optic neuropathy
Recently, an animal model for anterior ischaemic
optic neuropathy (AION) in rats and mice was
developed by Bernstein et al. In this model, a
photosensitizing agent is injected intravenously
into anaesthetized rats.
Using a custom-designed fundus contact lens,
a laser beam directly activates the dye within
the small vessels perfusing the optic nerve
while sparing the large calibre vessels
perfusing the inner retina. Pale oedema of the
optic nerve appears shortly thereafter.
Electrophysiologically, there is a decrease in
amplitude and, histologically, there is
impairment in axonal transport. The mean
RGC loss by 39 days after the treatment is
40%. A similar model was developed for use in
Inclusion Criteria for Clinical Trials
1.Subjects must be capable of providing informed
2.Subjects must be able to comply with the
3.Disease severity must be sufficient to
demonstrate a statistically significant and
clinically meaningful effect of therapy.
4.Specific diagnostic criteria must be defined to
ensure homogeneity of disease status, which can
lead to a more precise study.
5.Subjects must be capable of responding to the
proposed mechanism of action of the intervention
to be studied.
Exclusion Criteria for Clinical Trials
1.Subjects have concurrent disease that could
confound the response to therapy.
2.Subjects are unlikely to comply with the protocol
or likely to be lost to follow-up.
3.Subjects have known hypersensitivity or
intolerance to the proposed therapy.
4.Subjects use concomitant therapy that affects
either tear function or ocular surface integrity.
5.Subjects have had surgical or other manipulation
of the eye that could confound the outcome
parameters or interfere with the mechanism of
action of the proposed intervention to be studied.
FACILITATE MULTICENTER AND INTERNATIONAL
COLLABORATIVE CLINICAL TRIALS
The Subcommittee recommends the development
of criteria to be used in multinational venues.
Important aspects to consider for such international
trials are the use of uniform terminology. This may
require that terms are translated and back-
translated for clarity and accuracy It is necessary to
resolve cultural or ethnic connotations or
implications in terminology. There should be
uniform interpretation of outcome variables with
standardized protocols for measurement and
recording of data.
Testing procedures should be uniform, with use of
standardized reagents, standardized protocols,
and consistent recording of results. It is
necessary to maintain skill levels of data
collectors and observers, including certification of
investigators and research coordinators and
technicians. Attempts should be made to reduce
biases related to population differences (race,