Brimonidine Blocks Glutamate Excitotoxicity-Induced Oxidative Stress and Preserves Mitochondrial Transcription Factor A in Ischemic Retinal Injury
et al. (2012) Brimonidine Blocks Glutamate Excitotoxicity-Induced Oxidative Stress and Preserves
Mitochondrial Transcription Factor A in Ischemic Retinal Injury. PLoS ONE 7(10): e47098. doi:10.1371/journal.pone.0047098
Brimonidine Blocks Glutamate Excitotoxicity-Induced Oxidative Stress and Preserves Mitochondrial Transcription Factor A in Ischemic Retinal Injury
Dongwook Lee 0
Keun-Young Kim 0
You Hyun Noh 0
Stephen Chai 0
James D. Lindsey 0
Mark H. Ellisman 0
Robert N. Weinreb 0
Won-Kyu Ju 0
Steven Barnes, Dalhousie University, Canada
0 1 The Sophie and Arthur Laboratory for Optic Nerve Biology, Hamilton Glaucoma Center and Department of Ophthalmology, University of California San Diego, La Jolla, California, United States of America, 2 Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute, Chonbuk National University Hospital , Jeonju, Jeonbuk , Republic of Korea, 3 Center for Research on Biological Systems, National Center for Microscopy and Imaging Research, and Department of Neuroscience, University of California San Diego , La Jolla, California , United States of America
Glutamate excitotoxicity-induced oxidative stress have been linked to mitochondrial dysfunction in retinal ischemia and optic neuropathies including glaucoma. Brimonindine (BMD), an alpha 2-adrenergic receptor agonist, contributes to the neuroprotection of retinal ganglion cells (RGCs) against glutamate excitotoxicity or oxidative stress. However, the molecular mechanisms of BMD-associated mitochondrial preservation in RGC protection against glutamate excitotoxicity-induced oxidative stress following retinal ischemic injury remain largely unknown. Here, we tested whether activation of alpha 2 adrenergic receptor by systemic BMD treatment blocks glutamate excitotoxicity-induced oxidative stress, and preserves the expression of mitochondrial transcription factor A (Tfam) and oxidative phosphorylation (OXPHOS) complex in ischemic retina. Sprague-Dawley rats received BMD (1 mg/kg/day) or vehicle (0.9% saline) systemically and then transient ischemia was induced by acute intraocular pressure elevation. Systemic BMD treatment significantly increased RGC survival at 4 weeks after ischemia. At 24 hours, BMD significantly decreased Bax expression but increased Bcl-xL and phosphorylated Bad protein expression in ischemic retina. Importantly. BMD significantly blocked the upregulations of N-methyl-D-aspartate receptors 1 and 2A protein expression, as well as of SOD2 protein expression in ischemic retina at 24 hours. During the early neurodegeneration following ischemic injury (12-72 hours), Tfam and OXPHOS complex protein expression were significantly increased in vehicle-treated retina. At 24 hours after ischemia, Tfam immunoreactivity was increased in the outer plexiform layer, inner nuclear layer, inner plexiform layer and ganglion cell layer. Further, Tfam protein was expressed predominantly in RGCs. Finally, BMD preserved Tfam immunoreactivity in RGCs as well as Tfam/OXPHOS complex protein expression in the retinal extracts against ischemic injury. Our findings suggest that systemic BMD treatment protects RGCs by blockade of glutamate excitotoxicity-induced oxidative stress and subsequent preservation of Tfam/OXPHOS complex expression in ischemic retina.
Funding: This work was supported in part by the National Institute of Health (NIH) grant EY018658 (WKJ), NCRR (National Center for Research Resources) P41
RR004050 (MHE), an unrestricted grant from Research to Prevent Blindness (New York, NY), and overseas research funds of Chonbuk National University in 2010
(DL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding
was received for this study.
Competing Interests: The authors have declared that no competing interests exist.
Glutamate excitotoxicity-induced oxidative stress has been
linked to mitochondrial dysfunction in retinal ischemia and optic
neuropathies including glaucoma , suggesting a distinct
mitochondrial dysfunction-mediated cell death pathway is
activated in retinal injury. Growing evidence indicates that glutamate
excitotoxicity and/or oxidative stress is associated with
mitochondrial DNA (mtDNA) damage-related mitochondrial dysfunction in
retinal neurodegeneration [1,5,7,8]. However, the molecular
mechanisms underlying these effects are poorly understood.
Brimonidine (BMD), a selective alpha 2-adrenergic agonist that
lowers intraocular pressure (IOP), protects retinal ganglion cells
(RGCs) against glutamate excitotoxicity in culture system in vitro
[9,10] as well as in rodent models of experimental ischemia and
glaucoma . Its potential mechanisms include inhibition of
glutamate release, upregulation of brain-derived neurotrophic
factor expression, regulation of cytosolic Ca2+ signaling, and
modulation of N-methyl-D-aspartate receptor (NMDAR, also as
known as NR) function [12,16,17]. In addition, BMD is
neuroprotective against oxidative stress that induces reactive
oxygen species (ROS) formation including superoxide radicals
(O22) in culture system in vitro  as well as in rodent models of
ocular hypertension [13,18,19]. Superoxide dismutases (SODs),
cytosolic SOD1 and mitochondrial SOD2, are expressed in the
ganglion cell layer (GCL) and inner plexiform layer (IPL) in rodent
retina . Emerging evidence suggests that SOD2 play a
proFigure 1. BMD treatment and induction of transient retinal ischemia. (A) Diagram for BMD administration before and after ischemic injury.
BMD or vehicle were injected by IP for short terms or administrated systemically for 4 weeks using osmotic pumps. (B) IOP elevation in the rat eyes
following transient ischemic injury. (B) Mean IOP was 76.164.2 mmHg during anterior chamber perfusion with saline. In contrast, mean IOP of
contralateral control eyes was 11.261.7 mmHg (n = 10). BMD, brimonidine.
tective role against neuronal cell death that induced by glutamate
excitotoxicity and oxidative stress .
Mitochondrial transcription factor A (Tfam, also as known as
mtTFA), a nucleus encoded DNA-binding protein in
mitochondria, has an important role in mitochondrial gene expression and
mtDNA maintenance, and therefore is essential for oxidative
phosphorylation (OXPHOS)-mediated ATP synthesis .
Mice lacking Tfam have impaired mtDNA transcription and loss of
mtDNA that leads to bioenergetics dysfunction and embryonic
lethality . In contrast, overexpression of Tfam mediates
delayed neuronal death following transient forebrain ischemia in
mice  as well as neonatal hypoxic-ischemic brain injury
rapidly increased Tfam and OXPHOS complex IV protein
expression in a rat model, suggesting these responses may support
endogenous repair mechanisms for mtDNA damage following
hypoxic-ischemic brain injury .
Here, we tested whether activation of alpha 2 adrenergic
receptor by systemic BMD treatment blocks glutamate
excitotoxicityinduced oxidative stress and preserves the expression of Tfam
and OXPHOS complex in ischemic retina.
Brimonidine Decreases Bax but Increases Bcl-xL and pBad
The systemic treatment with BMD or vehicle began 24 hours
before the induction of transient retinal ischemia and continued
for 4 weeks post-ischemia (Figure 1 A). In addition, the rats
received an IP injection of BMD or vehicle 24 hours prior to
ischemia induction and another injection during the procedure.
Transient retinal ischemia was induced by acute IOP elevation to
76.164.2 mmHg for 50 min during anterior chamber perfusion
with saline (n = 10; Figure 1 B). The pressure was enough to
Figure 2. BMD-mediated protection of RGC survival in ischemic injury. BMD or vehicle were administrated systemically for 4 weeks using
osmotic pumps. (AC) Brn3a whole-mount immunohistochemistry. High magnification showed representative images from the middle area of
retinas. In comparison with normal control retina (A), vehicle-treated ischemic retina showed greater RGC loss (B). In contrast, BMD significantly
increased RGC survival in ischemic retina (C). (D) The quantitative analysis of RGC loss. Values are mean 6 SD (n = 6 retinas/group). *Significant at
p,0.05 compared with non-ischemic contralateral control retina or vehicle-treated ischemic retina. BMD, brimonidine. Scale bar = 100 mm.
Figure 3. BMD-mediated blockade of apoptotic pathway. BMD or vehicle were injected once by IP at 24 hours before and at the time of initial
IOP elevation for 24 hours after ischemia. Vehicle-treated ischemic retina significantly increased Bax, Bcl-xL, and pBad protein expression. In contrast,
BMD significantly decreased Bax protein expression, but increased Bcl-xL and pBad protein expression compared with vehicle-treated ischemic retina.
Values are mean 6 SD (n = 4 retinas/group). *Significant at p,0.05 compared with non-ischemic contralateral control retina or vehicle-treated
induce retinal ischemia and the phenotype was similar to
pathologic acute angle closure glaucoma because when IOP
reached 60 mmHg, the retina flow rate decreased by 68% for
retinal artery in rat . The mean IOP of contralateral control
eyes was 11.261.7 mmHg (n = 9; Figure 1 B). The alpha
2adrenergic receptors are expressed in the somas of the cells in the
GCL and inner nuclear layer (INL) . To quantify RGC
survival following BMD treatment in ischemic retina, we
performed whole-mount immunohistochemistry for Brn3a
antibody. In comparison with control retina (Figure 2 A and D),
vehicle-treated ischemic retinas showed about 27% of RGC loss at
4 weeks after transient ischemia, (p,0.05; Figure 2 B and D). In
contrast, BMD treatment significantly increased RGC survival by
an approximate 20% compared to vehicle-treated ischemic retina
(p,0.05; Figure 2 C and D).
To determine whether BMD modulates apoptotic cell death
pathway in ischemic retina, we performed Western blot analysis
using antibodies for Bax, Bcl-xL and phosphorylated Bad (pBad).
We found that Bax protein expression was significantly increased
in vehicle-treated ischemic retina by 8.9461.96-fold compared
with control (p,0.05). In contrast, BMD treatment significantly
decreased Bax expression by 4.9262.24-fold in ischemic retina
(p,0.05; Figure 3). In comparison with control, vehicle-treated
ischemic retina significantly increased Bcl-xL and pBad protein
expression by 1.6660.14- and 4.4660.55-fold, respectively
(p,0.05; Figure 3). Intriguingly, BMD treatment showed greater
increases of Bcl-xL and pBad protein expression by 2.2960.1- and
8.461.86-fold in ischemic retina, respectively (p,0.05; Figure 3).
Brimonidine Blocks the Upregulation of GFAP, NMDA
Receptors and SOD2 Expression
We investigated whether BMD blocks the upregulations of glial
fibrillary acidic protein (GFAP), NR1 and NR2A, and SOD2
expression in ischemic retina. Astroglia and/or m uller cells
activation coincides with RGC degeneration in the hypertensive
retina of the human, rat or mouse [3,3133]. As shown in Figure 4,
vehicle-treated ischemic retina significantly increased GFAP
protein expression by 4.2060.30-fold compared with control. In
contrast, BMD treatment significantly decreased GFAP protein
expression in ischemic retina at 24 hours (p,0.05; Figure 4 A).
When the primary antibody was omitted, as a control for GFAP
immunohistochemistry, there was no labeling by the secondary
antibody in control retina (Figure 4 B). Compared with control
retina (Figure 4 C), GFAP immunoreactivity was increased in
mu ller cells and astrocytes of the nerve fiber layer of
vehicletreated ischemic retina at 24 hours (Figure 4 D). In contrast, BMD
treatment decreased GFAP immunoreactivity in ischemic retina
(Figure 4 E). NR1, NR2A and SOD2 protein expression were
significantly increased by 1.5960.13-, 1.9660.03-, and
Figure 4. BMD-mediated blockade of the upregulations of GFAP, NMDA receptors and SOD2 expression in ischemic retina. BMD or
vehicle were injected once by IP at 24 hours before and at the time of initial IOP elevation for 24 hours after ischemia. (A) Vehicle-treated ischemic
retina significantly increased GFAP protein expression compared with control retina. In contrast, BMD significantly decreased GFAP protein
expression in ischemic retina. (BE) GFAP immunohistochemisty. When the primary antibody for GFAP was omitted, there was no labeling of the
secondary antibody (B). In comparison with control retina (C), vehicle-treated ischemic retina showed activation of mu ller cells (arrowheads) and
astrocytes (D). In contrast, BMD significantly decreased activation of mu ller cells and astrocytes (E). ONL, outer nuclear layer; OPL, outer plexiform
layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale = 20 mm. (F) Vehicle-treated ischemic retina significantly
increased NR1, NR2A and SOD2 protein expression compared with control retina. In contrast, BMD significantly decreased NR1, NR2A and SOD2
protein expression in ischemic retina. Values are mean 6 SD (n = 4 retinas/group). *Significant at p,0.05 compared with non-ischemic contralateral
control retina or vehicle-treated ischemic retina.
1.5160.15-fold, respectively, compared with control retina
(p,0.05). In contrast, BMD treatment significantly decreased
NR1, NR2A and SOD2 protein expression in ischemic retina
(p,0.05; Figure 4 F).
Brimonidine Preserves Tfam and OXPHOS Complex
To determine whether ischemic injury alters protein expression
for GFAP, Tfam and OXPHOS complex, we performed Western
blot analysis. Increases in GFAP, Tfam, and OXPHOS complex
(II and IV) protein expression were maximal 24 hours later by
2.760.15-, 2.460.16-, 3.3360.3-, and 1.4260.1-fold in ischemic
retina, respectively (p,0.05; Figure 5). The relative concentration
of each of these proteins expression was less at 72 hours than at 24
hours after ischemia-reperfusion. In addition, OXPHOS complex
I protein was significantly increased up to 72 hours (p,0.05;
Figure 5). However, there was no difference in ATP synthase
protein expression. These results suggest that ischemic injury
triggers the upregulation of Tfam and OXPHOS complex protein
expression in the early neurodegenerative events in the retina.
BMD treatment preserved Tfam protein expression at 24 hours
(p,0.05; Figure 6 A) compared with vehicle-treated ischemic
retina. When the primary antibody was omitted, as a control for
Tfam immunohistochemistry, there was no labeling by the
secondary antibody in control retina (Figure 6 B). Compared with
control retina (Figure 6 C), Tfam immunoreactivity was increased
in the outer plexiform layer (OPL) and IPL, as well as in neurons
of the GCL in vehicle-treated ischemic retina at 24 hours (Figure 6
D). In contrast, Tfam expression in the OPL, IPL and GCL
(Figure 7 E) of BMD-treated ischemic retina was similar to control
retinas. To confirm whether RGCs express Tfam, RGCs were
coimmunostained with Tfam and Thy1.1, a specific marker for
RGCs. Tfam was expressed predominantly in GCL neurons that
were positive for the RGC protein Thy1.1 (Figure 6 F and G). In
addition, there was no significant difference of Tfam expression
between vehicle- and BMD-treated non-ischemic control retinas
(p = 0.5; Figure 6 H), suggesting that BMD did not directly affect
expression of Tfam protein in control. Further, Western blot
analyses showed that BMD treatment also preserved OXPHOS
complex (I, II, III and IV) protein expression (p,0.05; Figure 7).
However, there was no difference in ATP synthase protein
expression at 24 hours compared with vehicle-treated ischemic
retina (Figure 7).
Consistent with our results that BMD promoted RGC survival
against ischemic injury, we found that BMD significantly
decreased Bax but increased Bcl-xL and pBad protein expression in
ischemic retina. Bax is a pro-apoptotic member of the Bcl-2 family
that is essential in many pathways of apoptosis [34,35] as well as
directly interacts with the component forming the mitochondrial
permeability transition pore that allows proteins to escape from the
mitochondria into cytosol to initiate apoptosis [36,37,38]. Bax is
counteracted by Bcl-xL that forms heterodimers with
dephosphorylation of Bad, which inactivates Bcl-xL and pBad eliminates
this dimerization, which activates Bcl-xL [39,40]. BMD-mediated
activation of alpha 2 receptors has been reported to activate the
phosphatidyl inositol-3-kinase (PI3K) and protein kinase/Akt (Akt)
pathways that increase survival factors including Bcl-2 and Bcl-xL
. Further, Bcl-xL promotes mitochondrial adenine-nucleotide
exchange and prevents mitochondrial hyperpolarization by
maintaining mitochondrial membrane permeability [42,43].
Together with these findings, our results suggest that BMD promotes
RGC survival against mitochondrial damage-mediated apoptotic
pathway in ischemic injury by increasing Bcl-xL and pBad
expression. These results reflect the possibility that increased
BclxL and pBad expression may represent endogenous repair
mechanism against apoptotic pathway and BMD may contribute
to the blockade of Bax-mediated increase of mitochondrial
membrane permeability or the promotion of mitochondrial
homeostasis in ischemic retinal injury.
Glutamate excitotoxicity and/or oxidative stress has been linked
to mitochondrial dysfunction in many neurodegenerative disorders
including retinal ischemia and glaucoma [2,4447]. Growing
evidence indicates that BMD is neuroprotective against glutamate
excitotoxicity or oxidative stress in retinal ischemia and
experimental glaucoma [1214,19]. Importantly, it has been proposed
that BMD may protect RGCs by inhibiting glutamate release,
modulating NMDA receptor function or interfering free oxygen
radicals [12,14,19]. In the current study, our findings
demonstrated the first evidence that BMD significantly blocks the
upregulation of NR1 and NR2A protein expression in ischemic retina,
suggesting the possibility that inhibition of glutamate release by
BMD-mediated alpha 2-adrenergic receptor activation may block
the upregulation of NMDA receptor expression and ameliorate
glutamate excitotoxicity-mediated neurodegeneration in ischemic
retina. Because MK801, a NMDA receptor antagonist, activates
PI3K/Akt pathway, reduces Bad expression, and subsequently
protects RGCs in ischemic retina , our findings suggest that
BMD may contribute to RGC survival through activation of
prosurvival PI2K/Akt pathway by not only activation of alpha
2adrenergic receptor, but also blockade of glutamate excitotoxicity
in ischemic retina. Thus, the potential mechanisms underlying
these synergic effects of BMD on ischemic retina should be further
We observed that BMD blocks the upregulation of SOD2
protein expression in ischemic retina. Because overexpression of
SOD2 contributes to the reduction of mitochondrial superoxide
level, protection of mitochondrial morphology and functions, and
mitochondrial resistance against glutamate excitotoxicity-induced
oxidative cytotoxicity , our result indicates that increasing
SOD2 expression may play a critical role in the mitochondria as
a compensatory mechanism for protecting RGCs against
glutamate exicitotoxicity-induced oxidative stress in ischemic retina.
Moreover, BMD-mediated preservation of mitochondrial SOD2
Figure 5. Alterations of Tfam and OXPHOS complex protein expression in ischemic retina. BMD or vehicle were injected once by IP at 24
hours before and at the time of initial IOP elevation and administrated intraperitoneally for 72 hours after ischemia. Acute IOP elevation significantly
increased GFAP, Tfam and OXPHOS complex (I, II, and IV), but did not change OXPHOS complex III and ATP synthase protein expression in ischemic
retina. Values are mean 6 SD (n = 4 retinas/group). *Significant at p,0.05 compared with non-ischemic contralateral control retina. Cx, Complex.
expression may provide an important neuroprotective implication
for ameliorating oxidative stress in ischemic retina. In addition, we
found that BMD blocks increased GFAP expression in mu ller cells
in ischemic retina. Although further studies need to be
investigated, it is possible that BMD-mediated protection may
indirectly modulate glial reaction against pressure-induced
ischemic insults. Thus, we believe that BMD has therapeutic potentials
for treating glutamate excitotoxicity- and/or oxidative
stressmediated neurodegenerative disorders including retinal ischemia
Ischemic injury triggers mitochondrial dysfunction including
ROS, mtDNA damage, or OXPHOS impairment in the central
nervous system [29,49]. Although mtDNA is particularly
susceptible to glutamate excitotoxicity-induced oxidative stress , it is
unknown whether glutamate excitotoxicity-induced oxidative
stress can directly trigger mtDNA dysfunction in ischemic retina.
Tfam regulates mtDNA copy numbers in mammals and the levels
of Tfam correlate with the levels of mtDNA [25,50]. Emerging
evidence suggests that the Tfam and OXPHOS complex IV are
rapidly increased in the early neurodegenerative events of neonatal
hypoxic-ischemic brain injury, suggesting that Tfam may
contribute to endogenous repair mechanism of injured brain neurons
. Further, recent studies reported that overexpression of Tfam
protect mitochondria against bamyloid-induced oxidative
damage in human SH-SY5Y neuroblastoma cells and ameliorates
delayed neuronal cell death in the hippocampus following
transient forebrain ischemia in mice [26,27]. On the other hand,
in mice lacking Tfam there is impaired mtDNA transcription and
mtDNA loss is triggered, therefore leading to mitochondrial
bioenergetic dysfunction-mediated embryonic lethality .
However, it is unknown that whether ischemic injury alters
Tfam/OXPHOS complex protein expression in the retina and
whether BMD blocks this alteration of Tfam/OXPHOS protein
expression in ischemic retina.
In the current study, we observed that retinal ischemic injury
triggers significant increase of Tfam/OXPHOS complex protein
expression in the early neurodegenerative events (1272 hours)
and BMD preserves Tfam/OXPHOS complex (I-IV) protein
expression in ischemic retina. Consistent with these results, we also
found that Tfam immunoreactivity is preserved in RGCs to the
control level following ischemic injury. These results suggest that
increasing Tfam/OXPHOS complex expression may be an
important mitochondria-related compensatory response against
glutamate excitotoxicity-induced oxidative stress in ischemic
retina. Further, BMD-mediated preservation of Tfam/OXPHOS
complex may provide a potential mechanism for protecting RGCs
against mitochondrial dysfunction-mediated ischemic retinal
injury. Future studies will investigate the precise molecular
mechanism underlying BMD-mediated mtDNA preservation in
ischemic retina or other optic neuropathies including glaucoma.
These results provide the evidence that systemic BMD
treatment may contribute to RGC survival by blockade of
glutamate exicitotoxicity-induced oxidative stress in ischemic
retina. Moreover, the preservation of Tfam/OXPHOS complex
expression by BMD may provide an important therapeutic
potential for protecting RGCs against mitochondrial dysfunction
induced by glutamate excitotoxicity and/or oxidative stress in
ischemic retina. Thus, our findings suggest that the potential
mechanisms underlying these effects by BMD may provide new
therapeutic strategies for ameliorating mitochondrial
dysfunctionmediated neurodegeneration in ischemic injury.
Materials and Methods
Female, Sprague-Dawley rats (250300 g in weight; Harlan
Laboratories, Indianapolis, IN) were housed in covered cages, fed
with a standard rodent diet ad libitum, and kept on a 12 hours
light/12 hours dark cycle. All procedures concerning animals were
performed in accordance with the ARVO statement for the Use of
Animals in Ophthalmic and Vision Research and under protocols
approved by institutional IACUC committees at the University of
California San Diego.
Induction of Transient Retinal Ischemia
The rats were anesthetized with a mixture of ketamine (50 mg/
kg, Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and
xylazine (5 mg/kg, TranquiVed; Vedeco, Inc., St. Joseph, MO) by
intraperitoneal (IP) injection. Eyes were also treated with 1%
proparacaine drops. A 30-gauge needle was inserted into the
anterior chamber of right eye that was connected by flexible
tubing to a saline reservoir. By raising the reservoir, IOP was
elevated to 7080 mmHg for 50 minutes. Shame treatment was
performed in the contralateral eyes by the insertion of a needle in
the anterior chamber without saline injection. Retinal ischemia
was confirmed by observing whitening of the iris and loss of the
retina red reflex. IOP was measured with a tonometer (TonoLab;
Tiolatoy, Helsinki, Finland) during ischemia. Non-ischemic
contralateral control retinas were used as a control.
BMD was purchased from United States Biological
(USBiological, Swampscott, MA). Two groups of rats, which were
randomly divided, were studied after transient retinal ischemia:
one group was treated with vehicle (0.9% saline, n = 13 rats), and
the other group was treated with BMD (1 mg/kg/day, n = 13
rats). BMD or vehicle were injected once by IP at 24 hours before
and at the time of initial IOP elevation for short term experiments,
as well as administrated systemically for 4 weeks using osmotic
pumps (Alzet; Durect, Cupertino, CA) that were implanted
subcutaneously on the back at 24 hours before transient retinal
Twelve to 72 hours after acute IOP elevation, light adapted rats
were anesthetized with IP injection of mixture of ketamine/
Figure 7. BMD-mediated restoration of OXPHOS complex protein expression in ischemic retina. BMD or vehicle were injected once by IP
at 24 hours before and at the time of initial IOP elevation for 24 hours after ishcemia. Note that BMD significantly decreased OXPHOS complex (I, II, III
and IV) but did not change ATP synthase protein expression in ischemic retina. Values are mean 6 SD (n = 4 retinas/group). There was no significant
difference between vehicle- and BMD-treated non-ischemic contralateral control retinas. *Significant at P,0.05 compared with vehicle-treated
ischemic retina. Cx, Complex; BMD, brimonidine.
xylazine, as described, and then rats were killed by CO2
inhalation. The rats were then perfused transcardially with 0.9%
saline followed by 4% paraformaldehyde in 1X phosphate buffer
saline (PBS, pH 7.4). Both eyes enucleated and fixed in 4%
paraformaldehyde in PBS for 4 hours at 4uC. After several washes
in PBS, the retinas were dissected and then dehydrated through
graded ethanols and embedded in polyester wax, as described
previously . For Western blot analysis, dissected retinas were
Whole-mount Immunohistochemical Analysis
Retinas from enucleated eyes were dissected as flattened
wholemounts at 4 weeks after transient retinal ischemia. Retinas were
immersed in PBS containing 30% sucrose for 24 hours at 4uC.
The retinas were blocked in PBS containing 3% donkey serum,
1% bovine serum albumin, 1% fish gel and 0.1% triton X-100,
and incubated with polyclonal goat anti-Brn3a antibody (1:500;
Santa Cruz Biotechnology, Santa Cruz, CA) for 72 hours at 4uC.
After several wash steps, the retinas were incubated with the
secondary antibody, Alexa Fluor-568 donkey anti-goat IgG
antibody (Invitrogen) for 24 hours, and subsequently washed with
PBS. Images were captured with a spinning-disc confocal
microscope (Olympus America Inc., Center Valley, PA) equipped
with a high-precision closed loop XY stage and closed loop Z
control with commercial mosaic acquisition software
(MicroBrightField; MBF Bioscience Inc., Williston, VT). The microscope
is equipped with high-resolution high-sensitive CCD camera for
high-speed mosaic acquisition. To count RGCs, each retinal
quadrant was divided into three zones by central, middle, and
peripheral retina (one sixth, three sixths, and five sixths of the
retinal radius). RGC densities were measured in 16 distinct areas
of 0.344 mm2 (two areas at central, middle, and peripheral per
retinal quadrant) per condition by two investigators in a masked
fashion, and the scores were averaged.
Immunohistochemical staining of 7 mm wax sections of full
thickness retina were performed as previously described . Five
sections per wax block from each group were used for
immunohistochemical analysis. Primary antibodies were
monoclonal mouse anti-GFAP antibody (1:500; Sigma, St. Louis, MO),
polyclonal goat anti-Tfam antibody (1:100; Santa Cruz
Biotechnology) and monoclonal mouse anti-Thy1.1 antibody (Clone
OX-7, 1:500; Millipore, Billerica, MA). To prevent non-specific
background, tissues were incubated in 1% bovine serum albumin/
PBS for 1 hour at room temperature before incubation with the
primary antibodies for 16 hours at 4uC. After several wash steps,
the tissues were incubated with the secondary antibodies,
FITCconjugated donkey anti-goat IgG antibody (Invitrogen, Carlsbad,
CA), Alexa Fluor 568 dye-conjugated goat anti-mouse IgG
antibody (Invitrogen) or Alexa Fluor 488 dye-conjugated
antirabbit IgG antibody (1:100; Invitrogen) for 4 hours at 4uC and
subsequently washed with PBS. The sections were counterstained
with the nucleic acid stain Hoechst 33342 (Invitrogen) in PBS.
Images were acquired with confocal microscopy (Olympus
FluoView1000; Olympus, Tokyo, Japan).
Western Blot Analysis
The retinas were homogenized in a glass-Teflon Potter
homogenizer in lysis buffer (20 mM HEPES [pH 7.5],
10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA,
1 mM DTT, 0.5% CHAPS, and complete protease inhibitors;
Roche Biochemicals, Indianapolis, IN). Each sample (10 mg; n = 3
retinas/groups) was separated by PAGE and electrotransferred to
polyvinylidenedifluoride membrane. The membrane was blocked
with 5% nonfat dry milk and 0.1% Tween-20 in PBS, incubated
with monoclonal mouse anti-GFAP (1:3000; Sigma), goat
polyclonal anti-Tfam antibody (1:1000; Santa Cruz Biotechnology),
mouse monoclonal anti-total OXPHOS complex antibody
(containing a mixture of antibodies to COXI-IV and ATP synthase,
1:3000; Invitrogen), rabbit polyclonal anti-Bax antibody (1:500;
Santa Cruz Biotechnology), rabbit polyclonal anti-Bcl-xL antibody
(1:1000; Cell Signaling, Danvers, MA), mouse monoclonal
antiphosphorylated Bad (pBad, 1:2000; Cell Signaling), mouse
monoclonal anti-NR1 antibody (1:1000; BD Pharmingen, San Diego,
CA), rabbit polyclonal anti-NR2A antibody (1:500; Millipore),
rabbit polyclonal anti-voltage-dependent anion channel (VDAC)
antibody (Porin, 1:1000; Calbiochem, La Jolla, CA) and mouse
monoclonal anti-actin antibody (1:5000, Millipore, Billerics, MA).
After several washes in Tween/PBS, the membranes were
incubated with peroxidase-conjugated donkey anti-goat IgG
(1:5000: Bio-Rad, Hercules, CA, USA), goat anti-rabbit IgG
(1:5000; Bio-Rad) or goat anti-mouse IgG (1:5000; Bio-Rad), and
developed using chemiluminescence detection (ECL Plus; GE
Healthcare Bio-Science, Piscataway, NJ). The scanned film
Images were analysed by ImageJ (http://rsb.info.nih.gov/ij/)
and band densities were normalized to the band densities for
actin or porin.
Data were presented as the mean 6 SD. Comparison of two or
three experimental conditions was evaluated using the unpaired,
two-tailed Students t-test or one-way analysis of variance and the
Bonferroni t-test. p,0.05 was considered to be statistically
Conceived and designed the experiments: DL W-KJ. Performed the
experiments: DL K-YK YHN SC W-KJ. Analyzed the data: DL K-YK
YHN SC W-KJ. Contributed reagents/materials/analysis tools: JDL MHE
RNW W-KJ. Wrote the paper: DL W-KJ.
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