Early Ischemia/reperfusion Injury in the Rat Retina
Authors: Mei-Lan. Ko, Chau-Fong Chen, Pai-Huei Peng, Yi- Hao Peng
PII: S0014-4835(11)00216-8 DOI: 10.1016/j.exer.2011.07.003 Reference: YEXER 5781
To appear in: Experimental Eye Research
Received Date: 10 January 2011 Accepted Date: 6 July 2011
Please cite this article as: Ko, M.-L., Chen, C.-F., Peng, P.-H., Peng, H. Simvastatin Upregulates Bcl-2 Expression and Protects Retinal Neurons from Early Ischemia/reperfusion Injury in the Rat Retina, Experimental Eye Research (2011), doi: 10.1016/j.exer.2011.07.003
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I. Highlights
Simvastatin has been shown to enhance retinal ganglion cell (RGC) survival against
ischemia-reperfusion (IR) injury. > Simvastatin treatment increased RGC survival by
proximately 40%. > Simvastatin upregulated the expression of Bcl-2 in the retina as shown by
western blot analysis. > The expression of Bax was unaffected. > The mechanism of
simvastatin-mediated neuroprotection likely involves the apoptotic pathway.
List of referee
George C.Y. Chiou, Ph.D
Department of Neuroscience and Experimental Therapeutics Professor and Director
Institute of Ocular Pharmacology
Founding Editor, Journal of Ocular Pharmacology and Therapeutics e-mail:chiou@medicine.tamhsc.edu
Hsiao-Ming Chao MD. PhD
Department of Ophthalmology, Cheng Hsin General Hospital , Taipei, Taiwan, Republic of China
ox_drchao@yahoo.ca
Isenmann S
Neuroregeneration Laboratory, Department of Neurology, Friedrich-Schiller University, Erlanger Allee 101, D-07747 Jena, Germany.
stefan.isenmann@med.uni-jena.de
Takahiro Kawaji, MD, Ph.D
Department of Ophthalmology and Visual Science, Graduate School of Medical Sciences,
Kumamoto University, 1-1-1 Honjo,
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kawag@white.plala.or.jp
Robert Ritch MD
Professor, New York Ear and Eye Infirmary, New York Medical college, USA
ritchmd@earthlink.net
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TITLE PAGE
I. Title:
Simvastatin Upregulates Bcl-2 Expression and Protects Retinal Neurons from
Early Ischemia/reperfusion Injury in the Rat Retina
II. Authors names and affiliations:
Mei-Lan. KO1,2
aaddch@gmail.com
1Department of Ophthalmology, General Hsin-Chu Hospital, Hsinchu, Taiwan
Address: No 25, Alley 442, Section one, Chin- Kuo Road, Hisnchu , Taiwan
2Department of Biomedical Engineering and Environmental Sciences, National
Tsing Hua University, Taiwan
Address: No. 101, Section 2, Kuang-Fu Road, Hsinchu, Taiwan
Chau-Fong CHEN3
chfochen@ntu.edu.tw
3Department of Physiology, College of Medicine, National Taiwan University,
Taipei. Taiwan.
Address: No 1, Section 1, Zen-Ai Rd, Taipei, Taiwan
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Pai-Huei PENG4 ,
paihuei@gmail.com
4Department of Ophthalmology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei,
Taiwan.
Address: No. 95, Wen Chang Road, Shih Lin District, Taipei City, Taiwan
Yi- Hao PENG5,
5Division of Pulmonary and Critical Care Medicine, Department of Respiratory
Therapy, China Medical University Hospital,
Address: No. 2, Yuh-Der Road, Taichung, 404, Taiwan
III. Corresponding authors:
(Pai Huei Peng, and Yi Hao Peng are equal corresponding authors)
Pai-Huei PENG
Department of Ophthalmology, Shin Kong Wu Ho-Su Memorial Hospital, No. 95, Wen
Chang Road, Shih Lin District, Taipei City, Taiwan
(O): 886-2-28332211
(F): 886-2-28331111
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paihuei@gmail.com
and
Yi-Hao PENG
Division of Pulmonary and Critical Care Medicine, Department of Respiratory Therapy,
China Medical University Hospital, No. 2, Yuh-Der Road, Taichung, 404, Taiwan
(O):886-4-22052121-1553
16080@mail.cmuh.org.tw
IV. Abstract
Simvastatin has been shown to enhance the survival of retinal ganglion cells (RGCs)
following ischemia-reperfusion (IR) injury by mediating the expression of stress
proteins. The purpose of this study was to investigate the effect of simvastatin on
retinal neurons and the expression of apoptotic proteins in a rat IR model. Wistar rats
received intravitreal injection of simvastatin immediately after retinal reperfusion.
Retinal ischemia was induced by increasing intraocular pressure to 150 mmHg for 60
min. The number of viable RGCs was measured after retrograde labeling with
Fluoro-Gold. Ischemia-induced apoptotic cell death was studied using terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We found
that simvastatin treatment enhanced RGC survival after retinal ischemia by
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approximately 40% and decreased retinal neuronal apoptosis. Using western blot
analysis, we found that simvastatin upregulated the expression of Bcl-2 in the retina.
In contrast, the level of the protein Bax was unaffected by simvastatin treatment. Our
results suggest that RGC loss induced by retinal IR may be prevented by simvastatin
and that the mechanism underlying this process possibly involves an alteration in the
apoptotic pathway.
V. Highlights
Simvastatin has been shown to enhance retinal ganglion cell (RGC) survival against
ischemia-reperfusion (IR) injury. > Simvastatin treatment increased RGC survival by
proximately 40%. > Simvastatin upregulated the expression of Bcl-2 in the retina as
shown by western blot analysis. > The expression of Bax was unaffected. > The
mechanism of simvastatin-mediated neuroprotection likely involves the apoptotic
pathway.
VI. Keywords:
Retinal ganglion cell, Simvastatin, Bcl-2, Bax, ischemia/reperfusion
VII. Abbreviations
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retinal ganglion cell (RGC), ischemia-reperfusion (IR), Terminal deoxynucleotidyl
transferase-mediated dUTP nick end-labeling (TUNEL), 3-hydroxy- 3-methylglutaryl
coenzyme A (HMG-CoA), ganglion cell layer (GCL), inner nuclear layer (INL),
Tris-buffered saline (TBS), normal goat serum (NGS),
VIII. Acknowledgement
There is no commercial relationship existed in the form of financial support or
personal financial interest on the study. This work is supported by Grant No 9707
from General Hsin Chu hospital, Hsin Chu, ROC.
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1. Introduction
Compromised retinal circulation can lead to retinal thinning and the loss of
neurons, in particular the death of retinal ganglion cells (RGCs) (Hughes, 1991). The
complex biochemical processes that follow ischemia-reperfusion (IR) have been
widely investigated and include energy depletion, glutamate-mediated excitotoxicity
and subsequent calcium overload, oxidative stress, inflammation and eventual
cellular necrosis, apoptosis (Buchi, 1992; Osborne et al., 2004) and necroptosis
(Rosenbaum et al., 2010). Animal models of transient IR have been established for
mimicking clinical diseases such as central retinal artery occlusion and ischemic optic
neuropathy to elucidate the pathological mechanisms underlying IR and to help
develop feasible treatments.
Apoptosis, a programmed form of cell death following a destructive cascade, is
triggered by a number of death receptor signaling pathways (Qu et al., 2010).
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
(TUNEL)-stained cells in the retinal ganglion cell layer (GCL) and the inner nuclear
layer (INL) of the retina are present approximately 2 h following IR and reach their
peak at 24 h (Lam et al., 1999). In addition, the expression of certain genes that are
related to the apoptotic pathway is initiated secondary to IR (Nickells et al., 2008).
Studies regarding neuroprotectants that may help rescue retinal neurons from
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apoptosis by mediating the expression of genes that are associated with the
apoptotic pathway are ongoing (Asomugha et al., 2010; Shen et al., 2010).
Statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase, have been described extensively for the treatment of hyperlipidemia since
1987. However, their pleiotropic properties beyond cholesterol reduction, including
anti-inflammation (Miyahara et al., 2004), immunosuppression (Kwak et al., 2001),
cytoskeletal modulation (Song et al., 2005) and protection against certain
neurological diseases(Menge et al., 2005), have gained increasing attention over the
past decade. Honjo et al. first used pravastatin and cerivastatin to investigate their
neuroprotective ability following retinal IR injury. They demonstrated that, following
IR, statin treatment preserved the retinal histo-architecture and ameliorated RGC
apoptosis by inhibiting leukocyte–endothelium interactions, in particular through
nitric oxide produced by the endothelium (Honjo et al., 2002). Another statin,
pitavastatin, has been shown to protect RGCs after retinal IR by modulating
intercellular adhesion molecules (Kawaji et al., 2007). Simvastatin is one of the most
potent members of the statins and was first marketed in the early 1990s. The
protective action of simvastatin for RGCs has been demonstrated in various
experimental models. Intravitreal injection of simvastatin has been shown to enhance
RGC survival by 90 and 19% at 7 and 14 days, respectively, after optic nerve axotomy
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by promoting heat shock protein expression and Akt activation (Kretz et al., 2006).
The same researchers also applied four different statins by subcutaneous injection
after retinal ischemia and found that the rescue rates for RGCs were 18.8, 58.3, 29.6
and 57.7% for mevastatin, pravastatin, lovastatin and simvastatin, respectively.
Modulating stress protein expression played an important role in the neuroprotective
effect of simvastatin in their study (Schmeer et al., 2008). In contrast, in a rat model
of autoimmune optic neuritis, simvastatin oral therapy failed to increase RGC survival
or visual function as assessed by visual evoked potential tests.
In addition to the above pathways through which simvastatin exerts its
neuroprotective action, regulation of genes that are related to cell death and survival
has been observed in cortical neurons following simvastatin treatment (Franke et al.,
2007; Johnson-Anuna et al., 2007). Among these genes, Bcl-2 is critical in the
apoptotic process (Reed, 1994). The ratio of anti-apoptotic Bcl-2 and pro-apoptotic
Bax plays an important role in cell fate (i.e., survival or death) following an apoptotic
stimulus (Oltvai et al., 1993). However, whether simvastatin mediates the expression
of these factors in the retina has not yet been investigated. Accordingly, the aim of
this study was to examine the effects of simvastatin on RGCs in a rat model of retinal
IR, with particular emphasis on the expression of proteins that are related to the
apoptotic pathway.
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2. Material and Methods
2.1 Animals
Eight-to-ten-week-old female Wistar rats weighing 200-250 g were used in this
study. All animal experiments and animal care were conducted in accordance with the
International Guiding Principles for Animal Research adopted by the Laboratory
Animal Center, National Taiwan University. The rats were housed under a 12-h
light/12-h dark cycle. The experiments were conducted under general anesthesia
induced by an intraperitoneal injection of a mixture of ketamine (50 mg/kg; Pfizer,
New York, NY, USA) and xylazine (8 mg/kg; Sigma, St. Louis, MO, USA).
2.2 Transient Retinal Ischemia
Under anesthesia, the animals were placed on a stereotaxic frame, and their
core body temperature was maintained at 37°C with a heating pad throughout the
experiment. A 30-gauge needle that was connected to a saline bottle was inserted
into the anterior chamber of the left eye. Acute retinal ischemia was induced by
elevating the bottle to a height that produced an intraocular pressure (IOP) of 150
mmHg for 60 min. The corneal puncture site was sealed with glue to ensure that the
increased IOP was maintained. In eyes that were subjected to a sham IR treatment,
the procedure performed was the same as above but without the elevation of the
saline bottle.
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2.3 Drug Administration
The animals were randomly assigned to four groups: sham solvent, sham
simvastatin, retinal IR solvent and retinal IR simvastatin. The rats were treated
with simvastatin (1.6 μg/μl) or vehicle (solvent, a mixture of ethanol and NaOH) via
intravitreal injection immediately after reperfusion.
2.4 Retrograde Labeling of RGCs and Counting
The head of the anesthetized rat was immobilized in a stereotaxic frame. The
scalp was incised, and two small holes were drilled into the skull above each superior
colliculus (bilaterally 6 mm posterior to Bregma and 1.5 mm lateral to the midline).
Three microliters of 5% Fluoro-Gold (Sigma-Aldrich) in sterilized distilled water was
injected with a micropipette at various depths in both superior colliculi.
RGC counting was performed three days after IR or sham injury. Rats were
euthanized with an overdose of chlorohydrate (Kanto Chemical, Tokyo, Japan). After
enucleation, the retina was gently peeled off and placed in a well containing 0.1 M
phosphate-buffered saline (PBS; pH 7.4) and then fixed in 4% paraformaldehyde for 1
h. After rinsing three times with PBS, each retina was divided equally into four
quadrants, flat-mounted on slides, and examined under fluorescence microscopy.
Each retinal quadrant was divided into three zones: central, middle, and
peripheral, approximately 1, 2, and 3 mm from the optic disc, respectively.In each
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zone, six microscopic fields of 430 μm × 285 μmeach along the medial line were
counted. In total, 72 fields were counted for each entire retina. The average RGC
density was derived from the total number of RGCs, which was then divided by the
total area of each retina that was counted.
2.5 TUNEL staining method
We used the TUNEL assay to evaluate apoptosis of retinal neurons after IR
injury. The eyes were enucleated 24 h after IR injury, and the retina was dissected as
described above. The tissue was then fixed with 10% formaldehyde for 24 h. The
whole retina was divided into two parts through the optic disc and then dehydrated
and embedded in paraffin. Sagittal sections (5 μm in thickness) were cut through the
optic disc and mounted. The deparaffinized sections were treated with the In Situ
Cell Death Detection Kit (Roche Molecular Biochemicals, Indianapolis, IN, USA). This
kit is based on the binding of digoxigenin-dUTP to the 3’-OH end of DNA by terminal
deoxynucleotidyl transferase (TdT) followed by incubation with an anti-digoxigenin
antibody that is conjugated to peroxidase. The sections were examined under 40×
magnification. Six microscopic fields of each eye—three adjacent areas on both sides
of the optic nerve head (1 mm from the optic nerve head)—were used to count the
TUNEL-positive cells in the ganglion cell layer (GCL) and the inner nuclear layer (INL).
The average number of TUNEL-positive cells in these two layers per field was used
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for analysis.
2.6 Western Blotting Analysis
The detached retina was homogenized for 30 min on ice in buffer containing
proteinase inhibitors (Sigma-Aldrich). The samples were boiled for 5 min in a water
bath and then centrifuged at 14,000×g for 30 min at 4°C. A Bradford assay (BioRad,
Hercules, CA, USA) was used to measure the protein concentration. Retinal proteins
(30 μg per sample) were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) on 12% (w/v) acrylamide gels and electroblotted onto a
nitrocellulose membrane. The membrane was blocked with non-fat dry milk (5% w/v)
in Tris-buffered saline (TBS) for 1 h and then incubated with a primary antibody
against BAX (1:250 in 2% normal goat serum [NGS] in TBS; Stressgen Bioreagents,
Victoria, BC, Canada) or Bcl-2 (1:200 in 2% NGS in TBS; Stressgen Bioreagents,
Victoria, BC, Canada). After three washes with Tween-TBS (BioRad, Hercules, CA,
USA), the secondary antibody (biotinylated anti-mouse IgG, 1:1000 in 2% NGS;
Vector, Burlingame, CA, USA) was applied for 1 h at room temperature. β-actin was
used as a loading control. Labeled proteins were detected using an enhanced
chemiluminescence western blotting system (ELC; Amersham Pharmacia Biotech,
Piscataway, NJ, USA), and the signals were quantified using computer-assisted
densitometry (Gel Pro 3.1; Media Cybernetics, Bethesda, FL, USA). The band
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densities of each sample were normalized to the β-actin band.
2.7 Statistical Analysis
All of the data in the text and figures are presented as mean ± SEM. Statistical
analysis was performed using the Student’s t-test for paired comparisons between
groups. Differences with a p-value of less than 0.05 were considered statically
significant.
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3. Results
3.1 Evaluation of Retrograde Labeling of Retinal Ganglion Cells
RGCs that were labeled with Fluro-Gold in a retrograde manner were counted 3
days following retinal IR or sham surgery with or without simvastatin treatment. The
mean densities of RGCs in the sham + solvent and sham + simvastatin groups were
1373.1 ± 18.7 and 1338.4 ± 45.3 cells/mm2, respectively (n = 7 and 4, respectively; Fig.
1). Three days after the ischemic episode, the density of RGCs was 1096.9 ± 37.3
cells/mm2 in the IR + solvent group, a decrease of approximately 20% compared with
the sham + solvent group (p < 0.001; n = 8 per group). In the IR-induced eyes that
were treated with simvastatin, the mean density of RGCs was 1208.6 ± 40.3
cells/mm2 (approximately 88% of control), which was significantly higher than the
eyes from the IR + solvent group (p = 0.025; n = 8 per group).
3.2 The Effect of Simvastatin Treatment on Apoptosis in the Retina Following
Ischemic Injury
To further test whether simvastatin protects RGCs from apoptosis, TUNEL
staining was used to detect DNA fragmentation of cells undergoing apoptosis (Fig. 2;
n = 6 per group). Fig. 2A-D shows representative pictures of TUNEL staining from the
different groups. TUNEL-positive cells in the GCL and INL of the retinas 24 h after IR
were counted. Significantly more TUNEL stained cells were found in the GCL and INL
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following IR (16.0 ± 6.7 vs. 0.2 ± 0.4 cells/field; p < 0.0001 compared with the sham +
solvent group). In rats subjected to IR with simvastatin treatment, the number of
TUNEL-positive cells was 9.2 ± 1.2 cells/field (p = 0.006 compared with the IR +
solvent group).
3.3 Bcl-2 and Bax protein levels Following Retinal Ischemia-reperfusion
The temporal expression of the anti-apoptotic protein Bcl-2 and the
pro-apoptotic protein Bax following retinal IR was examined by western blot analysis.
No significant change in Bcl-2 protein levels in the retina was observed 8, 16 or 24 h
following reperfusion (n = 3 at each time point; Fig. 3A). On the other hand, an
approximately 3-fold increase in retinal Bax protein level was found at 8 and 24 h
after reperfusion (n = 4 at each time point; p = 0.042 and 0.044, respectively,
compared with the normal control retinas; Fig. 3B). There was no significant
difference in Bax expression 48 h after reperfusion in comparison with the normal
control.
3.4 Bcl-2 protein levels are Upregulated after Simvastatin Treatment
The time course of changes in Bcl-2 protein levels in the retina after simvastatin
administration is shown in Fig. 4 (n = 4 at each time point). The level of Bcl-2 protein
in the retina was increased at 4 h after simvastatin treatment and reached a peak at
24 h (p = 0.034). The level of expression then declined and no significance difference
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was found 48 h after administration of simvastatin.
Fig. 5 shows the effect of simvastatin on Bcl-2 protein levels 24 h after retinal IR
was induced (n = 4 in each group). In the sham groups, the eyes that received
simvastatin treatment had a significantly higher level of Bcl-2 protein than those that
received solvent (p = 0.018). After retinal IR, the simvastatin-treated eyes also
showed a significant increase in Bcl-2 protein levels compared with solvent-treated
eyes (p = 0.044).
3.5 Bax Protein Levels are not Affected by Simvastatin Treatment
The level of the pro-apoptotic protein Bax in rat retinas with or without
simvastatin treatment 24 h after IR is shown in Fig. 6 (n = 4 in each group). Retinal
ischemia drastically upregulated Bax protein expression at 24 h (p = 0.021 between
the sham + solvent and IR + solvent groups and p = 0.025 between the sham +
simvastatin and IR + simvastatin groups). No significant difference in Bax protein
levels was found after simvastatin treatment.
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4. Discussion
In this study, we investigated the effects of simvastatin on RGCs following a
transient ischemic challenge. Our results suggest that simvastatin treatment
prevented the death of a proportion of RGCs following IR insult. Furthermore, the
increase of Bcl-2 protein level and amelioration of apoptotic neuronal death appear
to be associated—at least partially—with the neuroprotective effects of simvastatin.
The cholesterol-independent neuroprotective actions conferred by statins involve
a variety of putative mechanisms, including anti-inflammatory, antioxidant,
anti-apoptotic and endothelial protection (Schmeer et al., 2007). Simvastatin is
hydrophobic, which means it can cross the blood-brain barrier and possibly the
blood-retinal barrier more easily than hydrophilic statins. The above properties and
its safety suggest the potential neuroprotective benefit of simvastatin for long-term
clinical use. Long-term oral statin use reduces the risk of open-angle glaucoma, in
particular among patients with cardiovascular and lipid disorders. Improving ocular
blood flow and/or enhancing aqueous outflow via inhibition of rho kinase activity
were proposed to play a role in this protection(McGwin et al., 2004). In a 3-year
cohort study, stabilization of normal tension glaucoma after taking simvastatin was
demonstrated (Leung et al., 2010). An additional ocular beneficiary of statins is their
ability to prevent myofibroblast transdifferentiantion in Tenon’s tissue. This effect
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might provide a promising strategy for inhibiting wound scarring after glaucoma
filtration surgery (Meyer-Ter-Vehn et al., 2008). On the other hand, contradictory
results regarding the protective effects of statins have been reported. For example,
statins failed to protect RGCs in a rat model of autoimmune optic neuritis (Sattler et
al., 2005), and an inhibition of remyelination by blocking cholesterol synthesis was
proposed as a possible reason for this lack of protection. Moreover, differences in the
immunomodulatory effect of statins in various strains of animals and in humans is
another concern (Sattler et al., 2005).
Programmed cell death, or apoptosis, has been identified in the retina following
various injuries, including ischemia (Buchi, 1992; Berkelaar et al., 1994; Quigley et al.,
1995; Barber et al., 1998). Following an apoptotic stimulus, the 21 kDa pro-apoptotic
protein Bax is translocated to the mitochondria. The subsequent opening of the
mitochondrial permeability transition pore and release of cytochrome c ultimately
leads to a unidirectional apoptotic cascade. Bcl-2 can counter the actions of Bax and
thereby prevent the cell from succumbing to apoptosis. Thus, the balance between
Bcl-2 and Bax leads a cell toward survival or death. Two theories have been proposed
to explain the switch of apoptosis. In the direct activation model, Bcl-2 Homology 3
(BH3)-only proteins are classified as activators (which bind directly to BAX and BAK
and drive their activation) or sensitizers (which bind only to the Bcl-2 homologs)
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(Letai et al., 2002). Because recent data challenge the first model, the indirect
activation model suggests that the BH3-only proteins induce apoptosis by engaging
the various pro-survival proteins that guard BAX and BAK (Willis et al., 2007).
Regarding the response of Bcl-2 family members to retinal IR, ischemia-induced
BAX expression is increased, as demonstrated at both the mRNA and protein level
(Kaneda et al., 1999; Zhang et al., 2002; Ju et al., 2008). However, the biological
activity of Bcl-2 following retinal IR remains inconclusive (Kaneda et al., 1999; Ju et
al., 2008). A recent study unraveled the sequential regulation of Bcl-2 family
members in the ischemic retina (Produit-Zengaffinen et al., 2009). There was no
obvious change in Bcl-2 mRNA levels from 3 to 24 h after reperfusion, whereas the
protein level was significantly increased 3 h after reperfusion. Overall, the Bax/Bcl-2
balance was not affected in the initial phase following an ischemic challenge. Our
findings support a dysregulation of Bax protein levels and an unchanged Bcl-2 level
24 h after reperfusion.
Upregulation of Bcl-2 expression that gives RGCs protection from
ischemia-induced damage has been reported for an alpha-2 adrenoceptor agonist
(Lai et al., 2002). Moreover, our finding that simvastatin increases Bcl-2 levels in the
ischemic retina is consistent with similar findings in brain neurons. Johnson-Anuna et
al. found that simvastatin treatment resulted in increased Bcl-2 mRNA and protein
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levels and reduced cytotoxicity. Additionally, an antisense oligonucleotide directed
against Bcl-2 abolished simvastatin-induced Bcl-2 overexpression and
neuroprotection (Johnson-Anuna et al., 2007). In addition to an upregulation of Bcl-2
expression, a downregulation of Bax was observed in a rat model of Huntington’s
disease and in brain cells isolated from guinea pigs after challenge with the Bcl-2
inhibitor HA 14-1 or a nitric oxide donor (Franke et al., 2007; Patassini et al., 2008).
Despite our observation that simvastatin increased Bcl-2 upregulation in the retina,
Bax protein levels after simvastatin treatment were not reduced, as was seen in
brain neurons. This discrepancy might be a result of the different tissues studied
and/or the method of simvastatin delivery (intraperitoneal vs. intravitreal injection).
5. Conclusions
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Taken together, the level of the anti-apoptotic protein Bcl-2 was increased by
simvastatin in ischemic retinas. In contrast, the level of the pro-apoptotic protein Bax
was not affected by simvastatin treatment. Therefore, this increased Bcl-2/BAX ratio
might have contributed to decreased retinal neuron degeneration following ischemia.
Further studies exploring the long-term benefit of this treatment, as well as studies
detailing the mechanisms of its actions, are clearly needed.
ACKNOWLEDGEMENTS
The authors declare that there was no commercial financial support or personal
financial interest in this study. This work was supported by Grant No 9707 from
General Hsin Chu hospital, Hsin Chu, ROC.
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Fig. 1. Density of retinal ganglion cells (RGCs) treated with simvastatin or solvent in rats
subjected to retinal ischemia at 60 min. The number of viable RGCs is significantly decreased
following reperfusion at 3 days. Treatment with simvastatin partially inhibits
ischemia-induced RGC loss. Data are expressed as mean ± SEM. IR, ischemia-reperfusion; Sim,
simvastatin. N = 7, 4, 8, 8, in sham + solvent, sham + Sim, IR + solvent, and IR + Sim group,
respectively. * p < 0.05, *** p < 0.001.
Fig. 2. Simvastatin treatment inhibits retinal neuron apoptosis after ischemic injury. (A-D)
Representative photomicrographs showing TUNEL-labeled cells in retinal sections from
various treatment groups. The labeled nuclei were visualized as brown granules by
streptavidin-DAB staining and then counterstained with 1% Methyl Green. Barely visible
TUNEL-reactive cells in the retinas of the sham + solvent (A) and sham + simvastatin (B)
groups. (C) A robust increase of TUNEL positive cells is noticed in the ganglion cell layer (GCL)
and the inner nuclear layer (INL). (D) Treatment with simvastatin significantly reduces TUNEL
positive cells in both the GCL and INL. (E) Quantitation of TUNEL-labeled cells in each
treatment group. Data are expressed as mean ± SEM. IR, ischemia-reperfusion; Sim,
simvastatin. N = 6 in each group. ** p < 0.01, *** p < 0.001.
Fig. 3. Western blot analysis of Bcl-2 and Bax protein expression after retinal ischemia. (A)
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No detectable change in Bcl-2 expression is observed following reperfusion at 8 to 24 hours.
N = 3 at each time point. (B) Retina ischemia results in significant upregulation of Bax protein
expression at 8 and 24 hours following reperfusion. Data are expressed as mean ± SEM. C:
normal control retinas. N = 4 at each time point. * p < 0.05 when compared with the normal
control retinas.
Fig. 4. Western blot analysis showing the temporal pattern of protein expression of Bcl-2
after simvastatin treatment. (A) Representative blots of two samples. (B) Twenty-four hours
following simvastatin administration the protein expression of Bcl-2 in the retina is
significantly upregulated. Data are expressed as mean ± SEM. C: normal control retinas. N = 4
at each time point. * p < 0.05 when compared with the normal control retinas.
Fig. 5. Protein expression of Bcl-2 at 24 hours after retinal ischemia with or without
simvastatin treatment. (A) Upper panel shows the representative protein bands. (B)
Densitometry reveals a significant induction of Bcl-2 protein expression in the retinas of rats
treated with simvastatin after sham operation or retinal ischemia. Data are expressed as
mean ± SEM. IR, ischemia-reperfusion; Sim, simvastatin. N = 4 in each group. * p < 0.05.
Fig. 6 There is no effect of simvastatin on Bax protein expression in the retina after ischemia
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at 24 hours. Data are expressed as mean ± SEM. IR, ischemia-reperfusion; Sim, simvastatin. N
= 4 in each group. ** p < 0.01.
