Abstract
purpose. To develop and characterize a model of pressure-induced optic
neuropathy in rats.
methods. Experimental glaucoma was induced unilaterally in 174 Wistar rats,
using a diode laser with wavelength of 532 nm aimed at the trabecular
meshwork and episcleral veins (combination treatment group) or only at
the trabecular meshwork (trabecular group) through the external limbus.
Intraocular pressure (IOP) was measured by a tonometer in rats under
ketamine-xylazine anesthesia. Possible retinal vascular compromise was
evaluated by repeated fundus examinations and by histology. The degree
of retinal ganglion cell (RGC) loss was assessed by a masked,
semiautomated counting of optic nerve axons. Effects of laser treatment
on anterior ocular structures and retina were judged by light
microscopy.
results. After the laser treatment, IOP was increased in all eyes to higher than
the normal mean IOP of 19.4 ± 2.1 mm Hg (270 eyes). Peak IOP was
49.0 ± 6.1 mm Hg (n = 108) in the combination
group that was treated by a laser setting of 0.7 seconds and 0.4 W and
34.0 ± 5.7 mm Hg (n = 46) in the trabecular group.
Mean IOP after 6 weeks was 25.5 ± 2.9 mm Hg in glaucomatous eyes
in the combination group compared with 22.0 ± 1.8 mm Hg in the
trabecular group. IOP in the glaucomatous eyes was typically higher
than in the control eyes for at least 3 weeks. In the combination
group, RGC loss was 16.1% ± 14.4% at 1 week (n = 8, P = 0.01), 59.7% ± 25.7% at 6 weeks (n= 88, P < 0.001), and 70.9% ± 23.6% at
9 weeks (n = 12, P < 0.001). The
trabecular group had mean axonal loss of 19.1% ± 14.0% at 3 weeks
(n = 9, P = 0.004) and 24.3% ± 20.2%
at 6 weeks (n = 25, P < 0.001),
increasing to 48.4% ± 32.8% at 9 weeks (n = 12, P < 0.001). Laser treatment led to closure of
intertrabecular spaces and the major outflow channel. The retina and
choroid were normal by ophthalmoscopy at all times after treatment.
Light microscopic examination showed only loss of RGCs and their nerve
fibers.
conclusions. Increased IOP caused by a laser injury to the trabecular meshwork
represents a useful and efficient model of experimental glaucoma in
rats.
Glaucoma is the second leading cause of blindness
worldwide.
1 2 The pathophysiology of glaucoma and its
optimal treatment are still under investigation, although it is widely
accepted that the level of intraocular pressure (IOP) is a consistent
risk factor in its incidence, severity, and progression. Moreover, the
main approach to therapy for glaucoma is IOP
reduction.
1 3 4
Little is known about the nuclear and cytoplasmic signaling pathways
that are involved in degeneration of retinal ganglion cells (RGCs) in
glaucoma. Previous investigations have shown that RGCs die by apoptosis
in glaucomatous eyes of humans, monkeys, and rats.
5 6 7 The
stages leading to apoptosis and potential inhibitors of this process
are of broad interest among neuroscientists.
8 9 10 The
elucidation of apoptotic pathways in glaucoma may suggest
neuroprotective therapies other than IOP reduction.
11 12 13 The study of mechanisms and the molecular basis of glaucoma will be
enhanced by reproducible, efficient animal models.
Because the level of IOP is a major risk factor in human
glaucoma,
14 15 one experimental approach has been to
increase IOP to a level that preferentially kills RGCs without causing
ancillary injury to the retina and ocular structures. Both induced and
spontaneous increases in IOP simulate important aspects of RGC loss in
nonhuman primates, rats, rabbits, mice, and dogs. Cioffi and
Sullivan
16 have taken an alternative approach, inducing
vasoconstriction in posterior orbital blood vessels by prolonged
pharmacologic exposure.
16 Experimental IOP elevation in
monkeys using laser application was suggested by Gaasterland and
Kupfer
17 and later refined.
18 Although the
monkey model of laser-induced IOP increase is robust and has been used
by many laboratories, it has become too expensive for investigations
that require large numbers of animals to test mechanisms of RGC death
and its prevention.
17 19 The only reported methods for
measuring IOP in the mouse involve invasive
cannulation,
20 21 and methods for increasing IOP in the
rat have therefore been investigated. Johnson et al.
22 and
Morrison et al.
23 increased rat IOP by hyperosmotic saline
microinjection into limbal veins, calibrating the tonometer (Tonopen
XL; Mentor, Norwell, MA) to measure IOP.
24 Shareef et
al.
25 and others
26 27 cauterized large veins
draining the anterior rat eye and used the pneumatonograph to estimate
IOP. WoldeMussie and Feldman
28 and Wijono and
Ruiz
29 treated the limbal vessels of rat eyes with a laser
to decrease outflow, whereas Ueda et al.
30 enhanced the
laser uptake by ink injections into the anterior chamber before
treatment.
We have attempted to produce experimental IOP elevation in more than
100 rats with each of the four approaches just described. Some of them
produced elevated IOP and optic nerve damage, but the consistency of
IOP elevation and the ease of production of these models were not ideal
in our experience. As a result, we modified the delivery of laser
energy to the anterior structures of the rat eye to develop a reliable
model of glaucoma in rats. As described in the current report, our
model has its own advantages and weaknesses. We present herein
extensive observations on how variation in laser delivery affects the
height and duration of IOP increase. In addition, we demonstrate the
RGC loss pattern over time and the effect of laser treatment on the
outflow channels of the rat by histologic examination.
Rats were killed by exsanguination under deep ketamine-xylazine
anesthesia. They were perfused through the heart briefly with 4%
paraformaldehyde followed by 5% glutaraldehyde in 0.1 phosphate buffer
(pH 7.2), and the eyes and attached orbital optic nerves were removed.
A cross section of the optic nerve from both experimental and control
eyes was removed 1.5 mm posterior to the globe and postfixed in 1%
osmium tetroxide in phosphate buffer. Nerves were processed into epoxy
resin, sectioned at 1 μm, and stained with 1% toluidine blue.
The area of the optic nerve cross-section was measured by outlining its
outer border at ×10 magnification on an image analysis system (Sensys
digital camera and Metamorph software; Universal Imaging Corp., West
Chester, PA). Three area measurements were taken and the mean was used.
To measure the density and fiber diameter distributions, we captured
images with a ×100 phase-contrast objective from 10 randomly spaced
nerve regions. These were edited to eliminate non-neural objects, and
the size of each axon internal to its myelin sheath (minimum diameter)
and the density of axons per square millimeter were calculated for each
image and for the entire nerve. The mean density was multiplied by
nerve area to yield fiber number for each nerve. The total axon number
in the glaucomatous eye was compared with the control fellow eye to
yield a percentage loss value. The number of axons counted among the 10
images of each nerve was approximately 20% of the total optic nerve
area. The counting process was performed by observers masked to the
protocol used in each nerve.
According to our counting system, normal Wistar rats eyes have a
mean axon count of 87,318 ± 4,955. The 95% confidence limit for
normal axonal number is thus 11% of the mean count. We can say that a
difference greater than 11% is indicative of loss, with a probability
of 97.5%. The percentage loss of optic nerve axons in the glaucomatous
eyes increased significantly over time in groups 1 and 4 (
Fig. 4 ;
Table 2 ). Groups 2 and 3 were studied only at 9 weeks after treatment,
and the time course of damage therefore could not be presented. Eyes
treated with a combination treatment and laser setting of 0.7 seconds
and 0.4 W (group 1) had more damage than eyes with similar laser
settings limited only to the TM (group 4). In group 1, the mean axonal
damage at 1 week was 16.1% ± 14.4% (
n = 8,
P = 0.01, paired
t-test), with six of eight eyes showing axonal
loss (using the definition above). Mean axonal loss increased to 59.7%±
25.7% (
n = 88,
P = < 0.001, paired
t-test) at 6 weeks and to 70.9% ± 23.6% (
n = 12,
P = < 0.001, paired
t-test) at 9 weeks.
All treated eyes had axonal damage by 6 and 9 weeks after treatment.
The mean axonal loss with trabecular treatment (group 4) was milder
than that in group 1: 19.1% ± 14.0% at 3 weeks (n = 9, P = 0.004, paired t-test), 24.3% ± 20.2% at 6
weeks (n = 25, P < 0.001, paired t-test), and 48.4% ± 32.8% at 9 weeks (n = 12, P < 0.001, paired t-test). Eight of 9 eyes
in the 3-week group, 23 of 25 at the 6-week group, and 11 of 12 eyes in
the 9-week group had axonal loss.
The mean axonal loss with combination treatment and laser setting of
0.5 seconds and 0.6 W was significant at 9 weeks after laser 49.7% ±
24.0% (n = 12, P < 0.001, paired t-test). In contrast, the mean axonal loss for group 3,
which was treated by deliveries directed at the limbal and radial
episcleral plexus, but no deliveries to the TM, was not significant:
4.6% ± 16.2% (n = 8, P = 0.4, paired t-test).
There was a statistically significant correlation between the amount of
optic nerve damage and three assessments of IOP exposure. The strongest
correlation was with the positive integral, our estimate of cumulative
IOP elevation (
P < 0.0001,
r 2 = 0.40, linear regression;
Fig. 5A ). Damage was also related to peak IOP after treatment
(
P < 0.0001,
r 2 =
0.28, linear regression;
Fig. 5B ). The correlation between the amount
of damage and the maximal IOP difference between glaucomatous and
control eyes was also statistically significant (
P = 0.003,
r 2 = 0.16, linear regression;
Fig. 5C ).
Specific attention was given to examination of the outflow
channels, the ciliary body, the iris, and the episcleral area of 20
eyes of 10 laser-treated rats. Six rats were treated by a combination
treatment (group 1) and four rats were treated by trabecular treatment
(group 4), and all were killed 6 weeks after laser treatment.
The normal rat outflow area has some similarity to that of the human,
with trabecular beams that are covered by endothelial cells leading to
a large channel that is the equivalent of Schlemm’s canal.
Outflow vessels are occasionally seen connecting from this
channel to the episcleral vessels. The iris inserts posterior to this
trabecular area, and the ciliary body with its two layers of epithelium
is located on the peripheral iris, somewhat more anteriorly than in the
human eye.
In most of the laser-treated animals, there were clear abnormalities in
the outflow channels. These included loss of intertrabecular spaces,
compaction of trabecular beams, and partial or complete obliteration of
the Schlemm’s canal–like channel. In some areas, the iris was scarred
to the angle over the meshwork (peripheral anterior synechia). In two
treated eyes, there were red blood cells in the anterior chamber
remaining from hyphema. In the six rats in group 1, the ciliary body
was partially atrophied, whereas in the four rats in group 4 the gross
appearance of the ciliary body was normal
(Fig. 8) .
The ideal glaucoma model in rats should be simple to produce,
inexpensive, reproducible, and as similar to human glaucoma in its
effects as possible. We suggest that the translimbal photocoagulation
model in rats meets many of these criteria. The model produced elevated
IOP in nearly every treated eye. Six weeks after treatment, 50% to
60% of RGCs had died, and some RGC loss was present in most eyes. The
significant damage was confined to the RGC layer, nerve fiber layer,
and optic nerve axons. The outflow channels in the anterior chamber
were clearly abnormal. No clinical fundus abnormality was detected by
indirect ophthalmoscopy, despite IOP as high as 50 mm Hg. The mean
blood pressure of the rat is typically higher than 70 mm Hg (measured
under anesthesia by arterial cannulation in our acute experiments). In
the awake state, blood pressure may be higher. Thus, the IOP generated
by this model is unlikely to be high enough to occlude major retinal or
choroidal vessels. Consistent with this conclusion, we found no loss of
the midretinal architecture in the area supplied by retinal arteries
and no sign of choroidal infarction. Although the rat RGC and optic
nerve head structure differ substantially from the
human,
32 it appears that elevated IOP causes a selective
loss of RGCs, due at least in part to axonal injury at the area of the
optic nerve just at and behind the eye.
33
Groups 1, 2, and 4 showed significant optic nerve damage at 6 weeks.
Group 3, in which treatment was directed toward the vessels and not at
the TM, showed insignificant damage. In addition, animals treated by
acute elevation of IOP did not show any axonal damage. The laser
treatment of group 1 lead to the most significant optic nerve damage
associated with the highest mean and peak IOP. Damage to optic nerve
axons therefore was clearly related to IOP elevation, and more than a
brief, high increase in IOP was necessary to produce damage. This type
of treatment (group 1) may be used for investigating cellular processes
and mechanisms of RGC death in glaucoma, because it produced
substantial damage in a relatively short period. If the neuroprotective
effect of a new drug were to be investigated, it may be beneficial to
use the model with the laser setting used in group 4, because the
damage in these animals was substantial but occurred more slowly. This
would facilitate the detection of the effect of a proposed therapy
better than in eyes with rapid, severe optic nerve injury.
The translimbal photocoagulation technique is simple to perform, and a
large number of animals can be treated in 1 day. For those who are
familiar with slit lamp and laser use, the method could be learned in 1
hour with direct supervision. If an investigator were not familiar with
ophthalmic instrumentation, the learning time would be longer. However,
it requires access to a diode or similar continuous wave laser. In our
opinion, this method is simpler to perform than the saline injection or
the vein cautery methods, based on our personal experience with
performing each of these other methods in more than 150 rats each. The
saline injection model described by Morrison et
al.
23 requires extensive construction of fine glass
needles and tubing, as well as excellent microsurgical skills. The vein
cautery model
25 also requires a sterile approach, surgical
instruments, magnification through loupes or operating microscope, and
considerable experience to recognize the surgical landmarks and
variations in vein anatomy in the rat.
The expense of any rodent model is favorable compared with the use of
monkeys, yet even the rat models have substantial equipment,
facilities, and personnel costs. Our approach requires a laser costing
thousands of dollars. The saline and vein cautery
methods
21 23 require animal surgical facilities and
surgical instruments. Our method is performed by one investigator,
whereas the other methods are possible to perform alone, but are more
conveniently accomplished with an assistant, thus increasing the cost
of personnel.
A method that does not reliably produce increased IOP and optic nerve
damage has the obvious cost of wasted animals and the time and material
spent on animals in which no IOP elevation occurs. Our experience with
the saline and vein cautery models indicated that approximately half
the animals had IOP increases insufficient to produce RGC loss. This
led to a waste of half the time and expense for IOP measurement and
histologic counting of RGCs or axons. In contrast, one of the main
advantages of the translimbal photocoagulation model is that it is
effective at producing IOP elevation and damage in most animals.
In the laser model, mean IOP returned to baseline in many animals by 3
weeks after treatment. Although it would be ideal to have continued IOP
elevation over longer periods, 3 weeks of elevated IOP produces
substantial RGC loss, sufficient for most investigations. If longer IOP
elevation is desired, another laser treatment can be performed, but
this increases the occurrence of corneal decompensation. We were
concerned that all the injury to RGCs may have been due to very brief,
high IOP in the first day after laser treatment. To investigate this
possibility, we performed 6- to 8-hour IOP elevations. Because these
did not damage the RGCs, it is clear that chronic IOP elevation is
needed to cause damage. It can be argued that 3 weeks of elevated IOP
is not really a chronic model. Considering that the life span of a rat
is 2 years, 3 weeks of its life may be comparable to 2 years in the
life of a human who lives to be 80 years old.
We measured IOP in rats under ketamine-xylazine anesthesia with a
tonometer (Tonopen XL; Mentor) immediately after laser treatment, every
3 days for 2 weeks, and weekly thereafter. It would be ideal to have as
many measurements as possible. Jia et al.
34 trained Brown
Norway rats to allow daily tonometry without anesthesia. Unfortunately,
we have found that this is not possible with Wistar rats. General
anesthesia lowers IOP in the rat, but all experiments are performed by
comparing the IOP of the treated to the fellow, normal eye. Thus,
anesthesia affects the absolute, but not the relative, IOP difference.
IOP in rats varies in a circadian fashion, higher in the evening and at
dark than in the morning and in light.
35 36 We kept the
rats in a normal lighting rhythm to allow their normal IOP diurnal
pattern, reasoning that unknown effects may be caused by severe
alteration of the animals’ environment. Although Jia et
al.
36 found no damage to photoreceptors in rats
from constant light exposure, our albino rats may be more susceptible
to light toxicity.
Over time, corneal abnormalities developed in the treated eyes,
including dry eyes, opacities, and even atrophic ulcers. These
complications were also noted by us (unpublished observations, 2000) in
other glaucoma models in rats.
Of particular interest, we found that RGC loss continued after the
period of major IOP elevation. Possibly, neuronal death from primary
injury lags behind the initial event of IOP increase. Alternatively,
the continued loss of RGCs weeks after restoration of normal IOP may be
due to secondary degeneration. This process can be defined as death of
RGCs that survive the primary insult but are injured by toxic effects
of the primary degenerating neurons. Secondary degeneration has been
shown to occur in RGCs after partial optic nerve
transection.
37 38
We have shown that the amount of optic nerve damage is correlated with
the cumulative IOP exposure, as well as with peak IOP and maximal IOP
difference between the glaucoma and control eye. Johnson and
al.
39 have also found that optic nerve damage is linearly
correlated with IOP.
Previous studies have shown a preferential loss of large optic nerve
axons in human glaucomatous eyes and in monkey eyes with modest optic
nerve damage.
40 41 42 In the present investigation, there
was selectively greater loss of larger RGC axons at 1 week after
treatment, when damage was mild, but the selectivity was not still
present when three fourths of the nerve was gone. This is logical,
because larger axons make up a small proportion of the rat, monkey, and
human optic nerve. Clearly, after loss of the majority of axons, it
would be surprising to find any selectivity, because all RGC sizes
would have to be affected. We were somewhat surprised to find any
selectivity in this rat model, because the rat appears to have less
distinct segregation of RGCs by size, and because the damage was
produced quite rapidly, minimizing the chance of detecting subtle
differences in RGCs loss. In every investigation of human eyes with
glaucoma that has studied this question, selective loss of larger RGCs
was found at the stage of mild damage. It has been suggested that this
finding may be due to shrinkage of axons rather than selectivity;
however, there was no shift of the distribution of axonal diameter to
smaller sizes.
In summary, we have shown that translimbal laser photocoagulation to
the TM reliably causes sufficient IOP elevation to produce significant
optic nerve damage. Thus, this glaucoma model can be used to
investigate cellular events that lead to RGC death and to evaluate the
effect of new treatment strategies.
Supported in part by National Eye Institute Grants EY02120 (HAQ) and
EY01765 (Core Facility Grant, Wilmer Ophthalmological Institute) and by
the Glaucoma Research Foundation, San Francisco, California.
Submitted for publication May 24, 2001; revised September 7, 2001;
accepted October 18, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked“
advertisement” in accordance with 18 U.S.C. §1734
solely to indicate this fact.
Corresponding author: Harry A. Quigley, Wilmer 122, Johns Hopkins
Hospital, 600 North Wolfe Street, Baltimore, MD 21287;
[email protected].
| Group | Treatment Type | Deliveries to TM (n) | Deliveries to Episcleral Veins (n) | Laser Setting |
| 1 | Combination | 60–80 | 15–20 | 0.7 sec/0.4 W |
| 2 | Combination | 60–80 | 15–20 | 0.5 sec/0.6 W |
| 3* | Combination | 60–80 | 15–20 | 0.2 sec/1 W |
| 4 | Trabecular | 60–80 | 0 | 0.7 sec/0.4 W |
Table 2. Mean IOP, Peak IOP, and Optic Nerve Damage in the Treatment Groups
Table 2. Mean IOP, Peak IOP, and Optic Nerve Damage in the Treatment Groups
| Treatment Type | Eyes (n) | Mean IOP after 9 Weeks (mmHg ± SD) | | Peak IOP (mmHg ± SD) | | Axonal Loss (%) | |
| | Glaucoma | Control | Glaucoma | Control | 6 Weeks | 9 Weeks |
| Group 1: combination, 0.7 sec/0.4 W | 108 | 25.5 ± 2.9 | 19.8 ± 1.6 | 49.0 ± 6.1 | 27.7 ± 2.8 | 59.7 ± 25.7 | 70.9 ± 23.6 |
| Group 2: combination, 0.5 sec/0.6 W | 12 | 22.7 ± 3.4 | 19.5 ± 1.3 | 42.9 ± 4.0 | 26.2 ± 4.4 | NA | 49.7 ± 24.0 |
| Group 3: combination, 0.2 sec/1 W | 8 | 19.0 ± 4.2 | 19.3 ± 3.1 | 30.1 ± 6.9 | 25.8 ± 2.9 | NA | 4.6 ± 16.2 |
| Group 4: trabecular meshwork, 0.7 sec/0.4 W | 37 | 22.0 ± 1.8 | 19.2 ± 0.9 | 34.0 ± 5.7 | 24.5 ± 2.4 | 24.3 ± 20.2 | 48.4 ± 32.8 |
| Acute model (8 h) | 5 | 65 ± 5.6 | NR | 70 ± 2.3 | NR | −1.4 ± 10.8 | NA |
The authors acknowledge the original work on laser treatment to the
anterior segment of rats of Elisabeth WoldeMussie and thank her
for her generous collaboration in assisting us to modify her model.
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