Abstract
purpose. To examine the neuroprotective effect of theα 2-adrenergic agonist brimonidine in a chronic ocular
hypertension model.
methods. Intraocular pressure (IOP) was elevated by laser photocoagulation of
episcleral and limbal veins. Retinal ganglion cell loss was evaluated
in wholemounted retinas. Brimonidine or timolol was administered,
either at the time of or 10 days after IOP elevation and continued for
3 weeks. Drug-related immunohistochemical changes in glial fibrillary
acidic protein (GFAP) were also determined after 3 weeks.
results. Laser treatment caused a twofold IOP increase over baseline that was
maintained for 2 months. A time-dependent loss of ganglion cells
occurred with elevated IOP. Systemic administration of brimonidine or
timolol caused little decrease in IOP. After 3 weeks of elevated IOP,
ganglion cell loss in control rats was 33% ± 3%. Brimonidine reduced
the progressive loss of ganglion cells to 26% ± 1% and 15% ± 2%
at doses of 0.5 and 1 mg/kg · d, respectively. Timolol had no effect.
Ten days of high IOP resulted in 22% ± 4% ganglion cell loss.
Brimonidine administration initiated 10 days after IOP elevation
prevented any further loss of ganglion cells. In vehicle- or
timolol-treated rats, ganglion cell loss continued to 33%. The
increase in immunoreactivity of GFAP in ocular hypertensive retinas was
attenuated by brimonidine.
conclusions. Systemic application of brimonidine or timolol had little effect on
IOP. Brimonidine, but not timolol, showed significant protection of
retinal ganglion cells when applied at the time of IOP elevation and
prevented further cell loss when applied after IOP was elevated. This
indicates that brimonidine has a neuroprotective activity unrelated to
its effect on ocular hypotension.
Glaucoma-induced ganglion cell degeneration is one of the
leading causes of blindness. One of the most well-defined risk factors
of glaucomatous ganglion cell damage is chronic elevation of IOP.
Recent reports have hypothesized possible mechanisms of this
neurodegeneration. Increase in vitreal glutamate in humans with
glaucoma and animals with experimental hypertension has suggested that
glutamate excitotoxicity may be involved in glaucomatous retinal
damage.
1 Others have reported that reduction of transport
of neurotrophic factors may be the cause of ganglion cell
death.
2 3 In these situations, apoptosis has been reported
to be a process of ganglion cell death.
4 Such reports shed
light on the mechanisms of ganglion cell injury and suggest the need
for a direct therapeutic approach to prevent cell damage or enhance
survival.
The conventional treatment of glaucoma has been directed toward
controlling IOP. One of the compounds that are currently used in the
control of pressure for open-angle glaucoma is theα
2-adrenergic receptor agonist brimonidine. It
is very effective in lowering IOP. There is also evidence thatα
2-adrenergic agonists protect neurons from
damage in several models of ischemia. For example dexmedetomidine has
been reported to be protective in focal cerebral ischemia in rat and
rabbit
5 and in incomplete forebrain ischemia in
rats.
6 Brimonidine has also been shown to have
neuroprotective activity in a variety of neuronal injury models, such
as light-induced photoreceptor damage, optic nerve injury, and acute
retinal ischemia.
7 8 9 In the present study we established
laser-induced chronic ocular hypertension in rats and used this model
to investigate the neuroprotective effect of brimonidine. We compared
the neuroprotective effect of brimonidine to that of timolol, another
ocular hypotensive compound used in the treatment of glaucoma.
Experiments were conducted in strict accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research and
strict adherence to internal animal care and use committee guidelines.
Male Wistar rats weighing 350 to 450 g were used. IOP was elevated
by laser photocoagulation with a blue-green argon laser (Coherent, Palo
Alto, CA). Rats were anesthetized with a mixture of ketamine (50
mg/kg), acepromazine (1 mg/kg), and xylazine (25 mg/kg). Laser
treatment was performed on the episcleral veins within 0.5 to 0.8 mm
from the limbus and on the veins in the limbus. Treatment was applied
in two parts, 1 week apart. The amount of energy used was 1 W for 0.2
seconds, delivering a total of 130 to 150 spots (50–100 μm spot
size) with both laser treatments.
IOP was measured by tonometer (Tono-Pen; Mentor, Norwell, MA) as
described previously, with some modification.
10 Rats were
injected with 3.0 mg/kg acepromazine intramuscularly, enough to keep
them calm but not to sedate them. Proparacaine (0.5%) was applied
topically on the eyes to anesthetize the cornea. Fifteen readings were
taken and averaged to give one measurement. IOP measurements were taken
three times for the first 2 weeks and then once a week for the
remainder of the experimental period. In drug-treated rats, IOP was
measured once a week. To determine the correlation of elevated IOP and
ganglion cell loss, IOP was elevated for different periods ranging from
4 to 60 days, and ganglion cell loss was evaluated.
Drugs were administered continuously using an osmotic pump that
was inserted subcutaneously on the back. They were administered in two
different paradigms. In one group, brimonidine (0.5 or 1 mg/kg · d, n = 10), timolol (1 or 2 mg/kg · d, n = 10), or
phosphate-buffered saline (PBS) vehicle (n = 10) was
administered at the time of initial IOP elevation. Age-matched
naïve control rats (n = 5) were used to determine
baseline ganglion cell number. In another group, IOP was elevated for
10 days before drug treatment with 1 mg/kg · d brimonidine (n= 15) or 2 mg/kg · d timolol (n = 15), or PBS vehicle
(n = 15). As before, an age-matched group (n = 9)
was used to determine baseline. In both paradigms treatment was
continued for 3 weeks after drug administration.
Because the drug treatment was continuous for 3 weeks, it was important
to establish that the high dose of brimonidine did not cause sedation.
To determine this, rats were treated with brimonidine at 1 mg/kg · d
(n = 3) for 9 days, and locomotor activity was measured on
days 5 and 9 and compared with that of vehicle-treated rats. Activity
was measured in photocell cages where horizontal and vertical movements
were recorded with an activity monitor (Digiscan Animal Activity
Monitor; Omnitech Electronics, Columbus, OH). Locomotor activity was
defined as the total number of horizontal and vertical beam
interruptions over a 5-minute period.
The effect of elevated IOP on retinal ganglion cell loss was
examined histologically in wholemounted retinas. At the end of each
experimental period, the ganglion cells were labeled by retrograde
transport using crystals of 3000 molecular weight (MW) dextran
tetramethylrhodamine (DTMR; Molecular Probes, Eugene, OR). This dye
traveled by diffusion at the rapid rate of 2 mm/hour and did not
require active transport.
11 Thus, we assumed that all the
ganglion cells would be labeled regardless of health, as long as they
were present. Rats were deeply anesthetized and the optic nerve was
exposed. A longitudinal incision was made on the meningis, and the
optic nerve was completely sectioned at approximately 2 to 3 mm from
the globe. Crystals of DTMR were applied to the cut end of the optic
nerve. To determine whether IOP elevation impeded the transport of the
dye, we compared effectiveness of labeling in normotensive eyes
(
n = 5), with eyes in which IOP was raised twofold by laser
treatment for 1 day (
n = 5). Twenty-four hours after
application of DTMR, the rats were killed, eyes enucleated, and fixed
with 4% paraformaldehyde. The retinas were wholemounted, and labeled
ganglion cells in eight central (0.66–1.103 mm from the edge of the
optic disc) and four to eight peripheral (1.98–2.43 mm) areas in four
quadrants of the retina were counted under 400× magnification. These
areas represent 2.34% to 3.12% of the ganglion cells in the retina.
Non–laser-treated, age-matched retinas were counted in the same areas
to estimate total ganglion cell number. Ganglion cell loss in
experimental eyes was calculated as percentage of cell loss compared
with the non–laser-treated control. Percentage protection of ganglion
cells by test compounds was calculated by comparing to vehicle-treated
rats.
We had shown that elevation of IOP resulted in increase in
immunoreactivity of intermediate filament glial fibrillary acidic
protein (GFAP) in Müller cells.
12 In this study we
examined whether treatment with brimonidine affects the increase in
this protein. At the end of the experimental period, fixed retinas were
cryoprotected with sucrose (10%–30%) at 4°C for 2 days. Cuts were
made at the horizontal meridian, and retinas were frozen in optimal
cutting temperature compound (OCT; Tissue Tek, Torrance, CA). Cross
sections of 15 μm were made from the upper half of the retina,
collected on gelatin-coated slides, and air dried. The sections were
then rehydrated and incubated with 10% normal goat serum in PBS for 30
minutes to block nonspecific sites. The sections were incubated
overnight with the primary antibody; polyclonal rabbit anti-cow GFAP
(Dako, Carpinteria, CA) diluted at 1:500, at 4°C in a humidified
chamber. The following day they were rinsed with PBS and incubated with
biotinylated secondary antibody for 60 minutes followed by
indocarbocyanine (Cy 2)-conjugated avidin (Jackson ImmunoResearch, West
Grove, PA.), rinsed, and coverslipped. Control samples were incubated
without primary antibodies. Quantitative analysis of the immunostaining
was performed by measuring fluorescence intensity by computer (Image
Pro software; Media Cybernetics, Silver Spring, MD) using the“
count-measure” function. Several sections were made, and five to
six samples (every five sections) were immunostained. Images from these
sections from all the rats were captured on camera (Spot; Diagnostic
Instruments Inc., Sterling Heights, MI), with the same magnification
and exposure period on the same day, and stored in the computer for
further processing. Using the same size of sampling area in different
layers of the retina, fluorescence intensity was measured in pixels and
the numbers recorded. The absolute intensity values were normalized to
a mean value from non–laser-treated control retinas. The control value
was expressed as 1. Changes in intensity in laser-treated eyes were
calculated in relation to the normalized control.
The data are expressed as mean ± SEM. Statistical
comparison was made using unpaired Student’s t-test. P < 0.05 was considered significant.
Laser photocoagulation of limbal and episcleral veins performed
close to the limbus resulted in increased IOP. The baseline IOP in
acepromazine-treated non–laser-treated rats was 16 ± 0.4 mm Hg
(
n = 25). The first laser treatment increased IOP to
25 to 27 mm Hg, approximately 1.6-fold over baseline
(Table 1) . The second laser treatment increased IOP to a maximum of
approximately 32 to 35 mm Hg (approximately twofold). This level of IOP
was sustained for longer than 2 months, with a slight decrease.
Elevated IOP-induced ganglion cell loss was evaluated by labeling the
ganglion cells with DTMR at the optic nerve 1 day before the end of
each time point. To determine whether IOP elevation did not
obstruct the transport of the dye, we compared quantity of
labeling in normotensive eyes, with eyes in which IOP had been
increased twofold for 1 day. At normal IOP of 16.4 ± 0.1 mm Hg,
ganglion cell count was 1393 ± 47/mm
2 (
n = 5). At the high IOP of 30.2 ± 0.5 mm Hg, the
number of cells was 1207 ± 73/mm
2 (
n= 5). These numbers were not significantly different from each
other (
P > 0.05), indicating that the transport of
DTMR was not hindered by elevated IOP.
Elevation of IOP by laser treatment caused a time-related loss of
ganglion cells
(Table 1) . The loss of ganglion cells was linear for the
first 3 weeks, resulting in a calculated cell loss of 12% per week and
slowed to 2% per week from 3 weeks to 2 months. The average cell loss
over the 2-month period was at 5% per week. By examining the ganglion
cell loss in the different quadrants, we noted a regional variation in
which there were more cells lost in the superior half than in the
inferior half
(Table 2) . Although this difference was evident at all time points, it was
significant after 1 and 2 months of elevated IOP only.
The effect of timolol or brimonidine was evaluated after systemic
administration. These drugs have side effects, such as slowing of the
heart rate (timolol) and sedation and a decrease in blood pressure
(brimonidine), when administered systemically. Sedation was a big
concern, because it would interfere with the rats’ eating or drinking.
Therefore, we tested the highest dose (1 mg/kg · d) of brimonidine
for its sedative effect, using as a measure locomotor activity on days
5 and 9 after placing the osmotic pump. Total activity was 3360 ±
301 (vehicle) and 3159 ± 341 (brimonidine) on day 5
(P > 0.66) and 2177 ± 452 (vehicle) and
2511 ± 388 (brimonidine) on day 9 (P > 0.66).
These values were not significantly different from each other,
indicating that there was no side effect from this dose of brimonidine.
In addition, these animals appeared active when routinely observed in
their cages.
In the first treatment paradigm, the compounds were administered
immediately after initial laser treatment. Systemic administration of
either brimonidine or timolol lowered IOP very slightly by 5% to 10%
at both doses tested
(Fig. 1) . In the second treatment paradigm, in which drug treatment commenced
10 days after elevation of IOP, neither brimonidine nor timolol showed
any effect on IOP
(Fig. 2) .
After 3 weeks of elevated IOP, there was a considerable decrease
in labeled ganglion cells in retinas of vehicle-treated eyes compared
with those treated with brimonidine during the same period
(Fig. 3) . The ganglion cell loss, in vehicle-treated rats was 33% after 3
weeks of elevated IOP
(Fig. 4) . Brimonidine treatment during this 3-week period resulted in a
dose-related protection of ganglion cells in which cell loss was
reduced to 26% ± 1% and 16% ± 3% with 0.5- and 1-mg/kg · d
doses, respectively
(Fig. 4) . Timolol treatment did not have any
protective effect with either dose
(Fig. 4) .
After 10 days of IOP elevation there was a 22% ± 4% decrease in
ganglion cell number
(Fig. 5) . Drug treatment was initiated at this time and continued for 3 weeks.
Ganglion cell loss in brimonidine-treated rats remained at 22% ±
3.8%, whereas those treated with vehicle or timolol continued to 33%±
5.6% or 35% ± 3.3%, respectively
(Fig. 5) .
In normal retina, GFAP immunoreactivity is restricted to
astrocytes and end feet of the Müller cells at the inner limiting
membrane. However, its expression increases after different types of
retinal injury.
12 13 14 After elevation of IOP, GFAP
immunoreactivity increased throughout the length of the Müller
cells
(Fig. 6) . Treatment with brimonidine caused a significant decrease in GFAP
expression compared with vehicle-treated eyes
(Fig. 6) . This decrease
spanned the length of the Müller cells (from ganglion cell to
photoreceptor layers). Timolol did not show any decrease in
immunoreactivity of GFAP.
Glaucoma-induced vision loss is the result of degeneration of
ganglion cells and their axons. Although it is well established that
elevated IOP is one of the risk factors in this disease process the
mechanism of degeneration is unclear. The continued loss of vision in
eyes with glaucoma, despite control of IOP, emphasizes the need for
neuroprotective therapy. The development of a chronic ocular
hypertension model in the rat provides a valuable means of evaluating
the mechanism and process of injury and can be used to evaluate
compounds with neuroprotective activity.
4 10 15 16 17 18 In
this study, the neuroprotective effect of brimonidine was examined
using rats with laser-induced chronic ocular hypertension. All the rat
models reported by others and the one described in this study have
shown that elevation of IOP causes ganglion cell or optic nerve fiber
loss.
4 10 15 16 17 18 It has also been shown that the level of
IOP obtained determines the extent of ganglion cell loss.
4 In this study, IOP elevation of twofold caused ganglion cell loss of
44% after 2 months, averaging 5.5% per week. This result is
comparable to previous reports showing that cell losses of
approximately 37% and 47% were obtained after 6 and 10 weeks,
averaging 6% and 5% per week, respectively.
4 15 Other
investigators have observed cell loss at a slower rate of 1.4% per
week when the average cell loss was calculated over a longer period of
6 months and the increase in IOP was 1.6-fold or less.
17 We noted a biphasic rate of ganglion cell loss with a fast rate of 12%
per week for the first 3 weeks of IOP elevation, followed by a slower
rate of 2% per week for the remainder of the experimental period. The
rate of ganglion cell loss in the other studies is
linear.
4 15 17
In prior studies cautery was used once to occlude two or three major
episcleral veins, 2 to 3 mm from the limbus, whereas laser treatment in
the present study was performed twice, on several veins within 1 mm
from the limbus. Thus, in addition to a difference in the technique of
vessel occlusion, the discrepancy in rate of ganglion cell loss could
also be due to the rate at which the target IOP was reached and
maintained. In this study, target IOP was reached by two laser
treatments within 1 week, which may have initiated a faster rate of
initial ganglion cell loss. The consistent and significant loss of
ganglion cells after 3 weeks of elevated IOP provided us with an
opportunity to evaluate neuroprotective activity of pharmacologic
agents within a relatively short period.
In this study, we provide evidence that theα
2-adrenergic agonist brimonidine is
neuroprotective of ganglion cells in eyes with laser-induced chronic
ocular hypertension. Brimonidine is currently used to lower IOP in the
treatment of open-angle glaucoma.
19 20 21 The
neuroprotective effect of brimonidine was evaluated in two different
ways. When brimonidine was applied at the time of IOP elevation, before
pressure-induced ganglion cell injury began, it attenuated ganglion
cell loss by 50% (cell loss with vehicle 33% and with brimonidine
15%), in spite of the sustained increase in IOP. More interesting was
that when brimonidine was administered 10 days after IOP was elevated,
it prevented any further loss of ganglion cells. In both treatment
paradigms systemically applied brimonidine (400 μg/d or 17 μg/h to
each rat) reached the retina in sufficient amount to exert
neuroprotection but had little or no effect on IOP. However, topically
applied brimonidine (0.2%, 10 μg/5 μl) was effective in lowering
IOP by 40% (data not shown) and was neuroprotective of retinal neurons
in a rat acute retinal ischemia–reperfusion injury
model.
22 Moreover a recent study has shown that vitreal
levels of brimonidine after topical application of a 0.2% dose in
phakic eyes with planned vitrectomy was 9.3 ± 8
nM.
23 This concentration is more than adequate to activate
the α
2 receptors in the retina. The median
effective dose (EC
50) of brimonidine for
activating α
2 receptors is approximately 2
nM.
24
The neuroprotective effect of brimonidine was compared with that of
timolol. Both brimonidine and timolol are currently used for the
treatment of glaucoma, and they lower IOP significantly. It is thought
that lowering of IOP reduces injury and preserves the visual field. In
this study, brimonidine, but not timolol, was neuroprotective. Similar
to brimonidine, topical timolol (0.5%, 5 μl) caused a decrease in
IOP by 27% (data not shown). Similarly, the amount of timolol that
reached the eye when delivered by osmotic pump appeared to be lower
than required to decrease IOP. This emphasizes the direct
neuroprotective effect of brimonidine.
In several models of retinal injury, stress, or degeneration,
expression of GFAP increases in Müller
cells.
13 25 26 The increase in immunoreactivity of GFAP in
this ocular hypertension model may be a response to the stress and
pathologic process that leads to degeneration of ganglion cells. The
decrease in GFAP immunoreactivity by brimonidine suggests that
activation of the selective α
2-adrenergic
receptors reduces this injury in the retina.
Several mechanisms underlying the neuroprotective activity ofα
2-adrenergic agonists have been proposed.
Brimonidine has been shown to increase neurotrophic
factors
7 and thus may be neuroprotective by enhancing the
survival of ganglion cells in this hostile environment. In addition,
activation of presynaptic α
2-receptors results
in inhibition of transmitter release.
27 28 It is possible
that brimonidine treatment attenuated the release of glutamate in the
eyes with elevated IOP. Increase in vitreal glutamate has been
implicated in excitotoxicity of ganglion cells in
glaucoma.
29 Although we did not measure vitreal glutamate
in this study, there is a report showing that that brimonidine inhibits
accumulation of glutamate in the vitreous after acute retinal
ischemia.
9
In summary, we have shown that laser photocoagulation of limbal and
episcleral veins in the rat caused chronically elevated IOP, resulting
in significant loss of ganglion cells. Systemic treatment with theα 2 agonist brimonidine provided neuroprotection
to ganglion cells in this model.
The authors thank James Burke and Peter Baciu for critical comments
on the manuscript.