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Up-regulation of Brain-Derived Neurotrophic Factor Expression by Brimonidine in Rat Retinal Ganglion Cells
Hua Gao, MD, PhD;
Xiaoxi Qiao, MD, PhD;
Louis B. Cantor, MD;
Darrell WuDunn, MD, PhD
Arch Ophthalmol. 2002;120:797-803.
ABSTRACT
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Objectives Brimonidine tartrate ophth, an  2-adrenergic agonist,
is widely used as an antiglaucoma agent for lowering intraocular pressure.
Recent studies suggest that brimonidine may be neuroprotective for retinal
ganglion cells (RGCs) following optic nerve crush injury. Brain-derived neurotrophic
factor (BDNF), a potent neuroprotective factor present in the RGCs, promotes
RGC survival in culture and following optic nerve injury. We tested the hypothesis
that a possible mechanism of brimonidine neuroprotection is through up-regulation
of endogenous BDNF expression in the RGCs.
Methods A single dosage of brimonidine tartrate ophth solution (0.85-34µM)
was injected intravitreally into Sprague-Dawley rat eyes. The fellow eyes
of each animal were injected with balanced salt solution (BSS) and used as
control eyes. To determine BDNF messenger RNA expression, animal eyes were
enucleated and processed for in situ hybridization, or retinas were isolated
and processed for Northern blot analysis using rat BDNF radiolabeled riboprobes.
Results In the control eyes injected with saline, BDNF was present in a minority
of the RGCs. Two days after brimonidine injection, the number of BDNF-positive
RGCs was increased from 55% to 166%, depending on brimonidine concentrations,
when compared with those in the controls. In addition, the BDNF signal intensities
in individual RGCs were elevated 50% in brimonidine-injected eyes compared
with control eyes. Northern blot revealed a 28% increase of BDNF expression
in the brimonidine group compared with the controls (P
<.003). No significant difference was observed in BDNF receptor, trk B, expression between brimonidine, or BSS control groups.
Conclusions A single dose of a low concentration of intravitreal brimonidine is
sufficient to significantly increase endogenous BDNF expression in RGCs. These
results suggest that brimonidine neuroprotection may be mediated through up-regulation
of BDNF in the RGCs. The BDNF should be further investigated regarding its
role in the neuroprotective effects reported with brimonidine.
Clinical Relevance Brimonidine may be (potentially) used clinically as a neuroprotective
agent in optic neuropathy, including glaucoma, and ischemic and traumatic
optic neuropathy.
INTRODUCTION
GLAUCOMA IS a significant public health problem worldwide.1
The current treatment strategies, including medications and surgery, focus
exclusively on lowering the intraocular pressure (IOP), though IOP is just
one of the risk factors for glaucoma. Clinical observations and research studies
suggest other factors that may play important roles in the development of
glaucomatous optic neuropathy. Recent research in neuroscience provides some
clues on mechanisms of neuro-degeneration, which is improving the understanding
of the pathophysiology of glaucoma. Neuronal degeneration is caused by a variety
of primary insults to the neurons, such as mechanical trauma, ischemia, or
genetic susceptibility. These primary insults seem to initiate a cascade of
events that cause injured neurons to release noxious and degenerative substances,
which leads to secondary neuronal degeneration in surrounding cells. The final
common themes responsible for neuronal damage and eventual cell death by apoptosis
are apparently caused by loss of neurotrophic support or release of excitotoxins
such as glutamate. Although the causes of primary neuronal degeneration may
be unknown, neuronal degeneration could be halted or at least delayed if a
secondary degenerative process could be prevented or modified.2-5
For example, neuroscientists have been using N-methyl-D-aspartate
(NMDA) inhibitors to block glutamate effects on neurons, which can cause calcium
influx into neurons leading to cell death.6-8
Neuronal degeneration can be grouped into 2 categories depending on
the location of the primary insult—somogenic or axogenic. In somogenic
processes such as stroke, the neuronal cell body is damaged in the early course
of the disease. In axogenic disorders such as spinal cord injury, amyotrophic
lateral sclerosis, and probably glaucoma, the axon is damaged initially, leaving
the cell body viable for a much longer time.9
Thus, neuroprotective agents such as neurotrophic factors can be used to promote
neuronal survival and to delay cell death.
Brimonidine tartrate (hereafter referred to as brimondine) ophth is
an 2-adrenoreceptor agonist that lowers the intraocular
pressure (IOP) by reducing aqueous production and by increasing aqueous uveoscleral
outflows.10-13
Recent animal studies provide evidence that brimonidine has neuroprotective
properties in optic nerve degeneration, which is not related to its IOP-lowering
effects. Yoles et al14 studied the effects
of brimonidine on optic nerve degeneration in a rat model. They partially
crushed the rat optic nerve and immediately injected a single dose of brimonidine
or other 2-agonists into the intraperitoneal space. They
found a significant increase in the ganglion cell survival rate with 2-agonist intraperitoneal injection, as compared with the control of
saline injection. Brimonidine's effect was most significant—the loss
of ganglion cells 2 weeks after crush injury was 3 times lower in the brimonidine-treated
group than in the controls. They also found that brimonidine significantly
attenuated the decrease in compound action potential amplitude caused by crush
injury.14 Their results suggest that 2-agonists, especially brimonidine, may play neuroprotective roles by
delaying secondary neuronal degeneration in axogenic optic neuropathy caused
by mechanical injury. Wheeler et al15 reported
similar neuroprotective effects of brimonidine in a mechanically injured rat
optic nerve.15 Furthermore, they found that
topical brimonidine significantly delayed rat ganglion cell death caused by
acute retinal ischemia and reduced ganglion cell apoptosis as demonstrated
by the TUNEL (terminal deoxynucleotidyl transferase–mediated biotin-deoxyuridine
5-triphosphate nick-end labeling) staining techniques.15
What is the underlying mechanism by which brimonidine can promote ganglion
cell survival and protect them from degeneration caused by mechanical or ischemic
injuries? It is well known that brain-derived neurotrophic factor (BDNF) has
neuroprotective effects on retinal ganglion cells (RGCs). Brain-derived neurotrophic
factor is a potent neurotrophic factor that can promote survival and prevent
neuronal death after axotomy in the optic nerve16-19
or in a cell culture.20-22
Exogenous BDNF can enhance optic axon branching and remodeling in vivo23 and protect ganglion cells from retinal ischemic
injury.24 Brain-derived neurothophic factor
is present in the RGCs. Our previous study25
and others26 have shown that a subpopulation
of RGCs can express BDNF. Optic nerve crush injury can significantly elevate
BDNF expression in the RGCs.27
Since BDNF can promote ganglion cell survival, BDNF is expressed in
the RGCs, and brimonidine is neuroprotective for ganglion cells, a natural
question is whether brimonidine has any effect on BDNF expression, and whether
the neuroprotective effects of brimonidine are mediated, at least in part,
through regulation of BDNF expression. In the present study, brimonidine was
injected into rat eyes, and BDNF messenger RNA (mRNA) expression in the retina
was studied subsequently.
MATERIALS AND METHODS
ANIMALS
Six- to 7-week-old Sprague-Dawley albino rats (approximately 250 g)
were obtained from Charles River Laboratories (Wilmington, Mass). Animals
were fed ab libitum with Purina laboratory chow (Ralston Purina, Atlanta,
Ga) and water, with room lighting consisting of a 12-hour light/12-hour dark
cycle.
BRIMONIDINE INTRAVITREAL INJECTION
Animals were anesthetized with intraperitoneal injections of pentobarbital
sodium (Nembutal, Abbott Laboratories, North Chicago, Ill) (75 mg/kg). A 0.2%
brimonidine (3.4mM) ophthalmic solution (Allergan Inc, Irvine, Calif) was
serially diluted with balanced salt solution (BSS; Alcon Labs Inc, Forth Worth,
Tex) from 100-fold to 4000-fold (100-, 500-, 1000-, 2000-, 4000-fold, respectively)
to obtain final concentrations from 34µM to 0.85µM (34µM,
6.8µM, 3.4µM, 1.7µM, and 0.85µM, respectively). A
single dose of 5 µL of diluted brimonidine solutions was injected into
vitreous under a dissecting microscope, through a temporal postlimbus spot
using Hamilton microinjector (Hamilton Co, Reno, Nev). A 30-gauge needle was
first used to make a punch incision 0.5 mm posterior to the temporal limbus,
and a Hamilton needle was then inserted through the incision approximately
1.5 mm deep and angled toward the optic nerve until the tip of needle was
seen in the center of the vitreous. When the lens was occasionally involved,
a hard resistance could be felt, and the eye was discarded and not used for
the study. Since BSS was used to dilute brimonidine to obtain serial concentrations,
5 µL of BSS was used as a vehicle control and injected into the fellow
eyes. Animals were humanely killed 48 hours following injection. At least
3 animals were used for each concentration of brimonidine. Two rats were given
a brimonidine injection in only one eye, and the fellow eyes were not given
any injection and were processed for in situ hybridization. Five rats with
a 1.7µM brimonidine injection in one eye and a BSS injection in the
fellow eye, were humanely killed 1 week after injection, and eyes were processed
for Northern blot analysis.
TISSUE PREPARATION
Animals were humanely killed with overdose of pentobarbital. Eyes were
enucleated, an incision was made in the cornea, and eyes were fixed immediately
in 4% formaldehyde in 0.1M phosphate buffer (pH, 7.4). After 15 minutes in
the fixative, lenses were removed, and eyes were cut along the corneal optic
nerve axis into halves. Tissues were further fixed and cryoprotected overnight
in 4% formaldehyde, 0.5% glutaraldehyde, and 20% sucrose in 0.1M phosphate
buffer (pH, 7.4). Tissues were embedded in Tissue-Tek OCT compound (Miles
Inc, Naperville, Ill) and cryosectioned at a thickness of 10 µm at -21°C.
The brimonidine-injected and BSS-injected eye tissue sections were mounted
on the same slide and processed identically so that sections could be directly
compared, with as little processing variability as possible.
IN SITU HYBRIDIZATION
Standard protocols of riboprobe in situ hybridization were followed
as described in detail previously.27-28
Rat BDNF complementary DNA (cDNA) clone was obtained as a generous gift from
Genentech Inc (South San Francisco, Calif). It consists of 460 bases of coding
region and was inserted into plasmid pGEM-4Z.29
For the generation of antisense and sense BDNF riboprobes, the plasmid was
linearized with restriction enzymes EcoRI and HindIII, respectively.30
S-labeled antisense and sense riboprobes were transcribed using the Riboprobe
Gemini System according to manufacturer instructions (Promega, Madison, Wis).
The tissue sections were pretreated with 10 µg/mL of proteinase K at
37°C for 20 minutes, 0.25% acetic anhydrite, and 0.1M triethanolamide
for 10 minutes. The tissue sections were then incubated at 50°C on a slide
warmer for 18 ± 2 hours with the probe solutions containing 5 x
106 cpm/mL30 of S-labeled probes,
50% formamide, 10% dextran sulfate, 300mM sodium chloride, 0.5 mg/mL of transfer
RNA (tRNA), 10µM DTT, 0.02% Ficoll, 0.02% polyvinyl-pyrolidone, 0.02%
BSA, and 1mM EDTA in 10mM Tris-HCl (pH, 8.0). Following hybridization, the
slides were rinsed in 4x sodium citrate (SSC) (SSC: 150mM sodium chloride
and 15mM NaAc), digested with 20 µg/mL RNase A at 37°C for 30 minutes,
washed through descending concentrations of SSC to 0.1x SSC at 60°C
to 70°C. The slides were then dehydrated in ethanol, dried, and coated
with NTB-2 liquid emulsion (Kodak Inc, Rochester, NY). Following exposure
in the dark for 4 weeks, the emulsion was developed, and sections were stained
with hematoxylin-eosin.
Brain-derived neurotrophic factor receptor, trk
B, and mRNA expression were also examined in brimonidine- and BSS-injected
eyes. Trk B cDNA clone, a generous gift from the
Bristol-Myers Squibb Pharmaceutical Research Institute (Lawrenceville, NJ),
was in pGEM-3Z with an insertion of 432 base pairs (bp), encoding a portion
of the extracellular domain of mouse trk B receptor.
This clone was used to generate pan probe to detect all forms of trk B receptor.28 Restriction enzymes HindIII and BamHI were used to
linearize the plasmid for the generation of antisense and sense probes, respectively.30 S-labeled antisense and sense trk B riboprobes were transcribed using the Riboprobe Gemini System.
In situ hybridization was then performed as described previously.
IMAGE QUANTIFICATION
To determine and compare the numbers of BDNF-positive ganglion cells
in the retinas, cells were quantified using computer-enhanced video densitometry
(Southern Micro Instruments, Atlanta, Ga). Brain-derived neurotrophic factor
mRNA-positive cells were defined as those cells over which silver grains exceed
5 times the background value. Total cell number in the ganglion cell layer
was also counted and used as a denominator. Thus, the percentage of BDNF-positive
ganglion cells was determined. For each concentration of brimonidine, at least
3 animals were used and 3 tissue sections were counted for each animal eye.
Animal eyes injected with 1.7µM (2000-fold dilution) brimonidine
were used to determine and compare BDNF signal levels in individual ganglion
cells between groups, with and without brimonidine injection. Twenty to 30
BDNF-positive cells were randomly selected from each tissue section, and 3
sections were used from each animal. Silver grain densities over individual
BDNF-positive cells were determined using computerized densitometry as described
previously. Three animals were included for the brimonidine or BSS group.
A t test was used for statistical analysis between
the 2 groups.
NORTHERN BLOT
Two groups of rats were used for Northern blot analysis at 48 hours
after intravitreal injections (17 rats), and at 1 week after injections (5
rats). Brimonidine (1.7µM) was injected intravitreally in one eye of
each animal, and BSS in the fellow eyes. Animals were then humanely killed,
and retinas were dissected out and pooled in each group. Total retinal RNAs
were isolated as described previously.30 The
antisense BDNF RNA probe was synthesized as described previously using [phosphorus-32{32P} cytidine 5'-triphosphate. Northern blot analysis
was performed using standard methods: the total RNA of 30 µg was separated
on 1% agarose formaldehyde–denaturing gel. For the 1-week group, 10
µg of RNA was used. The RNA was blotted to 0.2 µm of neutral nylon
membranes (Schleicher and Schuell, Keene, NH) and hybridized to a 32P-labeled BDNF probe (3 x 106cpm/mL). The membrane
was then washed in graded SSC, dried, and exposed to a PhosphorImager plate
(Molecular Dynamics, Eugene, Ore). Relative abundance of mRNA was quantified
by reading the plate. Both bands of BDNF mRNA expression were used to perform
the densitometry. For accurate quantification, the same blot was stripped
off and hybridized to 32P-labeled  -actin probe. The ratio
of BDNF to actin densities was then used for comparison between the brimonidine
and BSS control groups. Northern analysis was repeated 5 times for the 48-hour
group, and 3 times for the 1-week group.
RESULTS
In the normal adult rat retina, BDNF mRNA expression is present in a
subpopulation of ganglion cells as described in our previous studies.25, 27 Brain-derived neurotrophic factor–positive
ganglion cells account for 5% to 6% of the cells in the ganglion cell layer
(GCL) and are randomly distributed throughout the retina. In the control eyes
injected with BSS in this study, in situ hybridization showed a very similar
image as in our prior study: 6.1% of ganglion cells are labeled positive with
the BDNF riboprobe (Figure 1A).
This demonstrates that the intravitreal injection used in the present study
does not alter the basal level of BDNF expression in the retina.
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Figure 1. In situ hybridization of brain-derived
neurotrophic factor (BDNF) messenger RNA (mRNA) expression in the retinas
of rat eyes injected intravitreally with a balanced salt solution (BSS) (A)
or brimonidine tartrate ophth solutions of 34µM, 6.8µM, 3.4µM,
1.7µM, and 0.85µM (B, C, D, E, and F, respectively). Arrows show
BDNF-positive ganglions in the ganglion cell layers. A, In retinas injected
with BSS, BDNF hybridization signals were present in a subpopulation of ganglion
cells. B-F, With brimonidine injections, the numbers of BDNF-positive ganglion
cells were significantly increased. Brain-derived neurotrophic factor signals
were also significantly more elevated in each of the individual cells in the
brimonidine injection group than those in the BSS control. GCL indicates ganglion
cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer. Scale
bar = 100 µm.
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Forty-eight hours following intravitreal injection of brimonidine, the
number of ganglion cells positive for BDNF significantly increased (Figure 1B-F). Quantification of the BDNF
signal revealed a significant increase in the number of BDNF-positive RGCs,
and in the intensity of silver grain density in each BDNF-positive RGC (Figure 2A, B). With a 34µM brimonidine
injection, 13.7% of ganglion cells are labeled for BDNF mRNA in the GCL—a
125% increase of BDNF-positive RGCs compared with the BSS control group (P = .014) (Figure 1B
and Figure 2A). When more diluted
brimonidine solutions were injected, the number of BDNF-positive RGCs continues
to be increased. As shown in Figure 2,
the number of BDNF-positive RGCs in the GCL reaches the peak when 6.8µM
brimonidine was injected (Figure 1C),
revealing a 166% increase of BDNF-positive cells compared with the BSS control
group (P = .003) (Figure 2A). With 3.4µM and 1.7µM brimonidine, the retinas
demonstrate 124% and 119% increases of BDNF cells in the GCL, respectively
(P = .012 and P = .006,
respectively) (Figure 1D-E and Figure 2A). Even when very low concentrations
of 0.85µM brimonidine were injected, there is still a 55% increase of
BDNF-positive RGCs in the retina when compared with the BSS controls (P = .01) (Figure 1F
and Figure 2A). Since the vitreous
volume in an adult rat is 56 µL ± 2 µL,31
and our intravitreal injection volume is 5 µL, the brimonidine solution
is further diluted approximately 12-fold in the vitreous. The final brimonidine
concentration ranged approximately from 72nM (4000-fold dilution) to 2833nM
(100-fold dilution). Thus, brimonidine induces BDNF expression in the ganglion
cells, probably through a very high affinity receptor.
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Figure 2. A, Percentage of BDNF-labeled
ganglion cells in the rat retinas with BSS or brimonidine tartrate ophth intravitreal
injections. There were significantly more BDNF-positive ganglion cells in
all brimonidine concentrations used than in the BSS controls. Specifically,
there were 125%, 166%, 124%, 119%, and 55% increases in 34µM, 6.8µM,
3.4µM, 1.7µM, and 0.85µM brimonidine injections, respectively,
with P = .01, P = .003, P = .01,
P = .006, and P = .01, respectively (mean
± SD number of rats used was at least 3 for each group). B, BDNF hybridization
signal intensity in individual ganglion cells of eyes injected with 1.7µM
brimonidine or BSS as control. Between 20 and 30 BDNF-labeled ganglion cells
were randomly selected from each of 3 tissue sections. Silver grain densities
over each individual cell were determined using a computerized densitometry
(see "Subjects, Materials, and Methods" section). Three animals were used
for the 1.7µM brimonidine or BSS group. A t test showed
a 50% increase of BDNF signal intensity in individual ganglion cells in the
brimonidine injection group compared with the BSS control group. See Figure
1 for abbreviation expansions.
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To confirm that elevated BDNF expression in the brimonidine group is
due not only to more BDNF-positive ganglion cells, but also to more expression
signals from individual ganglion cells, BDNF signals in individual ganglion
cells of eyes injected with 1.7µM brimonidine were quantified (see the
"Image Quantification" subsection of the "Subjects, Materials, and Methods"
section) and compared with the BSS control. A 50% increase of silver grains
was noticed in individual ganglion cells in the brimonidine group compared
with the BSS control group (P = .003) (Figure 2B). These results indicate that BDNF expression levels were
elevated in individual ganglion cells following brimonidine injection. Two
rats were given injections of 3.4µM of brimonidine in one eye, and the
fellow eyes were not injected, and used as natural controls. In situ hybridization
showed no difference in BDNF expression between these 2 natural control eyes
(data not presented) and eyes with BSS injection. This suggests that 3.4µM
of brimonidine intravitreal injection does not alter BDNF mRNA expression
in the contralateral eyes.
Although in situ hybridization is an excellent technique for detection
of the localization of mRNA signals, it is only a semiquantitative method
for signal quantification. To quantify the difference between brimonidine
and BSS control groups more accurately, Northern blot analysis was performed
using a rat BDNF riboprobe. Total RNAs were isolated from retinas of eyes
injected with either 1.7µM brimonidine or BSS. Northern blot revealed
2 characteristic bands migrating at 1.6 kilobases and 4.0 kilobases, corresponding
with the 2 forms of BDNF mRNA as reported in rodents (Figure 3A). This confirms that the signals detected in in situ hybridization
and in Northern blot analysis are indeed BDNF mRNA.28 -Actin
mRNA expression was also examined by reprobing the same blot and used as an
internal control. Densitometry analysis of the ratio of BDNF mRNA to -actin
mRNA signals shows a 28% increase of BDNF mRNA expression in the brimonidine
compared with that of the BSS control groups (P<.003)
(Figure 3B). One week after
a single 1.7µM brimonidine injection, there is still a 12% increase
of BDNF expression, as demonstrated by Northern blot analysis, but this is
not statistically significant (P = .21)
(Figure 3A, B). No difference was detected
in -actin expression between the brimonidine and BSS control groups
(Figure 3A), indicating that the
increase of BDNF mRNA expression is not due to an overall elevation of mRNA
expression in the retina. Thus, Northern blot analysis confirms the in situ
hybridization results and demonstrates that BDNF mRNA expression levels are
significantly elevated in rat retinas following intravitreal injection of
a very low (1.7µM) concentration of brimonidine solution.
We also examined trk B mRNA expression following
brimonidine injection. trk B is a BDNF receptor present
in the retina.32-33 In situ hybridization
demonstrated that trk B signals are located in the
GCL and inner nuclear layers (Figure 4),
which is very consistent with previous reports.32-33
Retinas of eyes injected with all concentrations of brimonidine were examined,
and image analysis indicated no significant difference of trk B mRNA expression between the brimonidine and BSS control groups.
Thus, brimonidine has no significant effect on trkB mRNA expression
in retinas 48 hours following intravitreal injection.
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Figure 4. In situ hybridization of trk B, a BDNF receptor, in rat retinas 2 days after brimonidine tartrate
ophth (A) or BSS (B) intravitreal injection. No significant difference was
detected between the 2 groups using image analysis (not shown). Arrows indicate
retinal cells positive for BDNF mRNA expression; GCL, ganglion cell layer;
INL, inner nuclear layer. Scale bar = 100 µm. See Figure 1 for abbreviation
expansions.
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COMMENT
The results of our studies clearly demonstrate that brimonidine up-regulates
BDNF mRNA expression in rat retinal ganglion cells. The BDNF mRNA expression
is elevated 28% 48 hours after a single dose of intravitreal brimonidine as
shown on Northern blot analysis (Figure 3). This elevation slowly declined to 12% 1 week after the injection
(P = .21), suggesting a pulse response of BDNF expression
to a single dose of brimonidine. The elevated BDNF expression was the result
of more BDNF-expressing ganglion cells and increased signal intensity in individual
cells after brimonidine injection (Figure
1 and Figure 2).
Intravitreal injection of brimonidine was used in our study. Questions
arise regarding whether topical brimonidine may cause the same effects (ie,
up-regulation of BDNF expression in the ganglion cells). A very recent study
by Kent et al34 showed that topical brimonidine
ophthalmic solutions (0.2%) administered to human eyes result in intravitreal
concentrations of 185nM ± 500nM.34 These
results match very well with the intravitreal brimonidine concentrations in
our study. Since the adult rat vitreous volume is 56 µL,31
the 5 µL of injected brimonidine solutions used in the present study
were diluted 12-fold in the rat vitreous. The actual intravitreal concentrations
of brimonidine were approximately 70nM, 140nM, 280nM, 560nM, and 2.8µM
for injected brimonidine solutions of 0.85µM, 1.7µM, 3.4µM,
6.8µM, and 34µM, respectively. In the Northern blot analysis,
1.7µM of brimonidine solution was used for intravitreal injection because
it resulted in a final intravitreal concentration of approximately 140nM,
which simulates the concentration achieved by topical brimonidine in the human
study.34 Since topical brimonidine can result
in similar intravitreal concentrations as in our intravitreal injections,
topical brimonidine may up-regulate BDNF expression in human retina. However,
different species may require different intravitreal concentrations of brimonidine
to elevate BDNF expression. Human retinal tissue is needed to examine BDNF
expression after brimonidine treatment.
Although brimonidine is a highly selective 2 agonist,
it can also bind to 1-receptors with low affinity and to
nonadrenergic imidazoline receptors with high affinity. Previous studies have
shown that brimonidine's effect has species differences: brimonidine stimulates
a central nervous system imidazoline receptor (Kd = 0.48nM) in monkeys to
decrease IOP, blood pressure, and heart rate; whereas in rabbits, it stimulates 2-receptors (Kd = 3.6nM) to decrease IOP.35
In the present study, only nanomolar levels of intravitreal concentrations
of brimonidine are needed to up-regulate BDNF expression. It is unlikely that
brimonidine acts on the 1-receptors. Brimonidine may act
on the 2-receptor or on the nonadrenergic imidazoline receptor.
In vitro cell cultures are needed determine which receptor(s) brimonidine
acts on to elevate BDNF expression.
Because it has been reported that as many as 40% to 50% of cells in
the GCL are displaced amacrine cells in the adult rat retina,36
an obvious question arises as to the type of cells involved with BDNF expression
in the GCL. If some BDNF-positive cells in the GCL were amacrine cells, some
amacrine cells in the inner nuclear layer should have also been labeled with
the BDNF probe, but this was not observed; no BDNF hybridization signal was
detected in the inner nuclear layer. Morphologically, BDNF-labeled cells in
the GCL are large and round, which is also consistent with ganglion cells.
Since BDNF can promote ganglion cell survival, it is reasonable to speculate
that promotion of ganglion cell survival by brimonidine in an optic nerve
injury model14 or ischemic model15
is mediated, at least in part, through BDNF up-regulation. It remains to be
studied how brimonidine up-regulates BDNF expression. Brain-derived neurotrophic
factor is a complex gene with 4 separate promoters located upstream of each
5' exon.37 Alternative usage of these
promoters and differential splicing result in 4 BDNF mRNAs with different
5' untranslated exons, which allow multiple points of BDNF mRNA regulation.38 Activation of the 2-receptor can
result in the regulation of multiple signaling pathways. Considering our results,
it is possible that some of the pathways hold potential interactions between
brimonidine and BDNF. For example, it has been demonstrated that 2-receptor stimulation induces the phosphorylation of mitogen-activated
protein kinase,39 while activation of this
kinase can lead to an increase of BDNF gene expression.40
In addition, 2-receptor agonists have been reported to induce
basic fibroblast growth factor expression in photoreceptors.41
Molecular mechanisms responsible for such interactions remain to be studied.
AUTHOR INFORMATION
Submitted for publication July 5, 2001; final revision received
November 16, 2001; accepted February 12, 2002.
Dr Cantor is a speaker at the Bureau of Alcon Labs Inc, Allergan Inc,
and Merck Co. He receives research support from Alcon Labs Inc, Allergan Inc,
Merck Co, Novartis AG, and Pharmacia Co. Both Drs Gao and Qiao contributed
equally to this study.
Corresponding author and reprints: Hua Gao, MD, PhD, Department of
Ophthalmology, Baylor College of Medicine, 6560 Fannin, Suite 2200, Houston,
TX 77030 (e-mail: huagao55{at}hotmail.com).
Department of Ophthalmology, Indiana University School of Medicine,
Indianapolis (Drs Gao, Cantor, and WuDunn), and the Department of Cellular
Biology and Anatomy, Louisiana State University Health Science Center, Shreveport,
La (Dr Qiao).
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