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Physiology and Pharmacology  |   July 2012
Methylene Blue Protects Primary Rat Retinal Ganglion Cells from Cellular Senescence
Author Notes
  • From the Department of Pharmacology & Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas. 
  • Corresponding author: Thomas Yorio, Department of Pharmacology & Neuroscience, UNT Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107; Thomas Yorio@UNTHSC.EDU
Investigative Ophthalmology & Visual Science July 2012, Vol.53, 4657-4667. doi:10.1167/iovs.12-9734
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      Donald R. Daudt, Brett Mueller, Yong H. Park, Yi Wen, Thomas Yorio; Methylene Blue Protects Primary Rat Retinal Ganglion Cells from Cellular Senescence. Invest. Ophthalmol. Vis. Sci. 2012;53(8):4657-4667. doi: 10.1167/iovs.12-9734.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Glaucoma is a progressive optic neuropathy characterized by loss of retinal ganglion cells (RGCs) and optic nerve degradation. Existing treatments focus on lowering IOP; however, vision loss may still progress. Neuroprotective drugs may be useful as an adjunct approach to prevent further loss of RGCs, although efficacious drugs are lacking. One agent, methylene blue, protects neurons during several neurodegenerative models. Methylene blue potentiates the electron transport chain by shuttling elections from NADH and FADH2 to coenzyme Q (CoQ) and cytochrome c. The purpose of this study was to determine if methylene blue could protect RGCs from noxious stimuli.

Methods.: Primary rat RGCs were isolated and cultured following a sequential immunopanning technique using P3-P7 Sprague-Dawley rats. Approximately 25,000 RGCs were seeded per coverslip and cultured for 3 days before testing. The RGCs were treated for 24 hours with rotenone or staurosporine or for 72 hours of hypoxia. Methylene blue was then assessed for protection of RGCs during each of these insults. Cell viability was measured using calcein Am and ethidium homodimer-1. Cytochrome c oxidase activity was measured using a cytochrome c oxidase assay kit to monitor the health of mitochondria.

Results.: Methylene blue (1 μM and 10 μM) significantly protected RGCs against 24 hours of 1 μM rotenone. Methylene blue (1 μM and 10 μM) significantly protected RGCs against 24 hours of treatment with 1 μM staurosporine and protected RGCs against 72 hours of hypoxia. Methylene blue increased cytochrome c oxidase activity in the presence of hydrogen peroxide.

Conclusions.: Methylene blue is a neuroprotective compound that can protect RGCs from toxic insults. Methylene blue's ability to increase cytochrome c oxidase and protect RGCs against these noxious stimuli supports its suggested mechanism of action, which is to preserve the electron transport chain. Further testing is needed to determine if methylene blue would be an efficacious treatment for the protection of neurodegeneration that occurs during optic neuropathy.

Introduction
Glaucoma is an optic neuropathy characterized by apoptosis of retinal ganglion cells (RGCs), cupping of the optic disc, and progressive deterioration of optic nerve axons resulting in impaired structure and function. 1,2 Glaucoma is the second leading cause of blindness worldwide. 1 Glaucoma is defined as the impairment of structure and function of the optic nerve, which results in death of the RGCs. 2 In primary open angle glaucoma, IOP is elevated, which leads to the pathology observed during this neurodegenerative disease. 1 Due to increased life span, the prevalence of glaucoma is projected to increase to 3.6 million by 2020. 3 Currently, treatments aim to increase aqueous humor outflow or decrease aqueous humor formation through surgical or drug intervention; however, vision loss may still progress in these patients. 4 The exact mechanisms causing loss of RGCs remains unknown; however, the axons forming the optic nerve become strained between lamina cribrosa cells, resulting in neurotrophin deprivation. 5,6 The lack of neurotrophin support leads to RGC apoptosis. 710  
Methylene blue (MB) is a potential neuroprotective intervention 11 that could be beneficial in protecting RGCs from death as a result of glaucoma. MB is already approved by the Food and Drug Administration to treat several ailments, including methemoglobinemia. 12 Chronic treatment with MB is safe and can protect against mental disorders and prevents encephalopathy in humans undergoing chemotherapy. 1316 MB is an autoxidizable phenothiazine with potent antioxidant activity with metabolic-enhancing properties. 17 MB has a high bioavailability in the brain and readily crosses the blood brain barrier. 18,19 Additionally, clinical trials are being performed with chronic MB treatment to prevent the progression of Alzheimer's disease. 20  
MB protects against several models of neurodegeneration in vivo, including amnestic mild cognition impairment, neurotoxin-induced impairment, and optic neuropathy. 2124 In rats, MB improves discrimination learning, facilitates the extinction of fear, and improves brain oxidative metabolism and memory retention. 2427 Whereas, optic neuropathy was induced in mice using rotenone, which causes mitochondrial dysfunction, 23 intravitreal injections of MB maintained visual acuity when compared with the control untreated contralateral eye. 23 MB also maintained structure and function of the retina during rotenone treatments. 23 Furthermore, MB preserved the integrity of the RGC layer. 24  
A number of neurodegenerative diseases are potentiated by mitochondrial dysfunction. This suggests that MB's electron coupling ability can potentially be protective to neuronal cells during neuronal disease states. 28 Investigating MB's protective properties during a model of RGC death could lead to a novel, inexpensive, and efficacious treatment of glaucoma. 
Materials and Methods
All animal procedures were performed in accordance and with the approval of the University of North Texas Health Science Center Institutional Animal Care and the ARVO Animal Use Committee guidelines. Female time-pregnant Sprague-Dawley rats were obtained from Charles River (Wilmington, MA). Primary rat RGCs were isolated and cultured following a sequential immunopanning technique using P3-P7 Sprague-Dawley rats. 29 RGCs were positively selected using a T11D7 (anti-thy1) antibody while macrophages were isolated using an antimacrophage antibody (CLAD51240; Cedarlane Laboratories, Hornby, ON). Primary RGCs were seeded 25,000 per coverslip, which were pretreated with poly-D-lysine (P6407; Sigma-Aldrich, St. Louis, MO) and mouse laminin (3400-010-01; Trevigen, Inc., Gaithersburg, MD). Cells were incubated at 37°C in 10% CO2 and air for all experiments, unless otherwise stated. 
Immunocytochemistry
Ben Barres trained our laboratory to perform his method of primary RGC isolation, which yields 99.7% ± 0.3% pure cultures of RGCs. 29 Primary RGCs were isolated and cultured for 7 days. Coverslips, containing RGCs, were washed three times with PBS followed by incubation with cold 100% methanol for 20 minutes. Cells were washed three times with PBS and then blocked using 5% Donkey serum in antibody buffer for 1 hour. Blocking solution was removed and cells were incubated with primary antibodies 1:100 anti-Thy 1.2 (550543; BD Pharmingen, San Diego, CA) and 1:100 anti—glial fibrillary acidic protein (GFAP) (SC-6170; Santa Cruz Biotechnology, Santa Cruz, CA). Cells were incubated overnight at 4°C. Cells were washed three times and then incubated with donkey or goat secondary antibodies for 2 hours at room temperature. Cells were washed three times using PBS and then mounted with 15 μL of prolong gold antifade reagent. Images were taken on a confocal laser scanning microscope 510 Meta (Carl Zeiss, Maple Grove, MN). 
Calcein-Acetomethoxy/Ethidium Homodimer-1 Cell-Survival Assay
Primary rat RGCs were cultured for 72 hours after isolation before subjection to any treatment. On completion of treatment, cell viability was determined using LIVE/DEAD Viability/Cytotoxicity Kit, ethidium homodimer-1, and calcein AM (L3224; Invitrogen, Carlsbad, CA). Cultured primary RGCs were washed three times with PBS. Cells were incubated in 2 μM calcein AM and 1 μM ethidium bromide homodimer-1 in PBS at 37°C for 20 minutes. Cells were washed three times in PBS and then mounted on slides containing fluorosave (345,789 EMD Biosciences, Gibbston, NJ). Fluorescence was measured on a Microphot FXA digital fluorescent microscope (Nikon, Melville, NY). An individual that was masked to the experimental design counted all cell survival assays. There were 25,000 primary RGCs seeded per coverslip. Cell survival was quantitated as amount of living cells (green) divided by the total amount of cells, living and dead (green + red). This ratio expresses the percentage of surviving cells from the specific insult. An average of 29 cells per image were quantified and five images were taken per coverslip, thus 145 primary RGCs were quantified per coverslip. 
Cytochrome c Oxidase Assay Kit
The Cytochrome c Oxidase Assay Kit (CYTOCOX1; Sigma-Aldrich, St. Louis, MO) was used to measure cytochrome c activity. The assay kit works by converting ferrocytochrome c to ferricytochrome c by cytochrome c oxidase. Ferrocytochrome c is measureable at 550 nm whereas ferricytochrome c is not. This means that a larger amount of functional cytochrome c oxidase will convert ferrocytochome c to ferricytochome c quicker than a smaller amount of cytochrome c oxidase. Thus, a larger amount of functional cytochrome c oxidase will change the absorbance at 550 nm quicker than less functional cytochrome c oxidase. RGCs were seeded 450,000 per well of a six-well dish. Primary RGCs were cultured for 7 days at 37°C at 10% C02 and air. Then the RGCs were treated with a vehicle, 500 μM hydrogen peroxide (216763; Sigma-Aldrich), MB (NDC 17478-504-10; Akorn, Lake Forest, IL), or both for 2 hours. Cells were removed using trypsin (T-9201; Sigma-Aldrich) and spun down at 1000 rpm for 5 minutes. Liquid was removed from pellet. Before each assay, photospectometer was blanked using 900 μL of assay buffer and 50 μL of suspended pellet. Then, 50 μL of ferrocytochrome c substrate solution was added and mixed through inversion. Assay measurements at 550 nm were taken every 10 seconds for 120 seconds. The amount of cytochrome c oxidase activity was expressed as a percent of control (vehicle) for each experimental group. 
Statistical Analysis
SigmaPlot 11.0 (Systat Software, Inc., San Jose, CA) was used to perform all statistical analyses. Results were expressed as mean ± SE. We used Mann-Whitney U test to test the difference between two groups. Significance was defined as a P value of 0.05 or less. 
Results
Primary Rat RGC Culture
Retinal tissue was collected from P3 to P7 Sprague Dawley rat pups. Macrophages were selected through an antimacrophage antibody and removed. RGCs were collected using the T11D7 (anti thy1) antibody. After 7 days in culture, the primary RGC cultures were incubated with a thy1.2 antibody (red) (Fig. 1A) and a GFAP antibody (green) (Fig. 1B). Virtually all cells were expressed the thy1.2 antigen, whereas none of the cells were GFAP positive. 
Figure 1. 
 
Primary rat RGC culture. Characterization of the isolated primary RGCs following Ben Barres' protocol. 29 (A) Cells were characterized by immunocytochemistry for normally expressed RGC marker Thy-1. (B) A blank of the same field was used. Thy-1 was detectable in virtually all of these cells. Scale bar, 100 μm.
Figure 1. 
 
Primary rat RGC culture. Characterization of the isolated primary RGCs following Ben Barres' protocol. 29 (A) Cells were characterized by immunocytochemistry for normally expressed RGC marker Thy-1. (B) A blank of the same field was used. Thy-1 was detectable in virtually all of these cells. Scale bar, 100 μm.
Rotenone Toxicity Concentration-Response
Rotenone induces mitochondrial dysfunction by inhibiting complex I of the electron transport chain. 30 Cell viability was determined using calcein AM/ethidium homodimmer-1 double staining (Fig. 2A). The vehicle-treated primary RGCs were set at 100% survival and used to standardize each rotenone treatment group. The addition of 100 nM rotenone for 24 hours left 59% of the RGCs viable (P < 0.001). The addition of 1 μM rotenone for 24 hours left 39% of RGCs viable (P < 0.001). The addition of 10 μM rotenone for 24 hours left 7% of RGCs viable (P < 0.001). The concentration 1 μM was used for all sequential rotenone experiments (Fig. 2B). 
Figure 2. 
 
Rotenone toxicity concentration-response. Rotenone significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning rotenone treatment. Cells were treated with or without varying concentrations of rotenone (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) Rotenone significantly killed primary RGCs at 100 nM (59% survival P < 0.001), 1 μM (39% survival P < 0.001), and 10 μM (7% survival P < 0.001) (n = 3).
Figure 2. 
 
Rotenone toxicity concentration-response. Rotenone significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning rotenone treatment. Cells were treated with or without varying concentrations of rotenone (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) Rotenone significantly killed primary RGCs at 100 nM (59% survival P < 0.001), 1 μM (39% survival P < 0.001), and 10 μM (7% survival P < 0.001) (n = 3).
MB Protects Primary Retinal Ganglion Cells against Rotenone Toxicity
Rotenone induces mitochondrial dysfunction by inhibiting complex I of the electron transport chain. 30 Cell viability was determined using calcein AM/ethidium homodimmer-1 double staining (Fig. 3A). Untreated cells were used as a control to quantitate the average amount of viable cells following primary RGC isolation and will be set at 100% survival for the experiments. Results are expressed as a ratio to the vehicle, 1 μM rotenone and (percentage of viable RGCs experimental group/percentage of viable RGCs vehicle group). The addition of 1 μM rotenone for 24 hours left 41% of RGCs viable. The addition of 100 nM MB did not induce a significant level of protection from 1 μM rotenone; however, 1 μM MB survival of the RGCs and 10 μM MB significantly protected against 1 μM rotenone toxicity (1.6-fold increase of viable cells, P < 0.001, and 1.6-fold increase of viable cells, P < 0.001) (Fig. 3B). Cells were not subjected to concentrations of MB over 10 μM due to toxic effects at higher doses. 31,32  
Figure 3. 
 
MB protects primary RGCs from rotenone toxicity. MB significantly protects primary RGCs from rotenone toxicity. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning rotenone treatment. Cells were treated with 1 μM rotenone and with or without varying concentrations of MB (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) A total of 1 μM rotenone significantly (41% survival P < 0.001) killed primary RGCs. MB significantly protected primary RGCs from rotenone toxicity at 1 μM (1.6-fold increase of RGC survival P < 0.001), and 10 μM (1.6-fold increase of RGC survival P < 0.001) (n = 6).
Figure 3. 
 
MB protects primary RGCs from rotenone toxicity. MB significantly protects primary RGCs from rotenone toxicity. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning rotenone treatment. Cells were treated with 1 μM rotenone and with or without varying concentrations of MB (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) A total of 1 μM rotenone significantly (41% survival P < 0.001) killed primary RGCs. MB significantly protected primary RGCs from rotenone toxicity at 1 μM (1.6-fold increase of RGC survival P < 0.001), and 10 μM (1.6-fold increase of RGC survival P < 0.001) (n = 6).
Staurosporine Toxicity Concentration-Response
Staurosporine is an apoptosis-inducing agent that was used to determine if MB can protect of primary RGCs against this cytotoxic insult. Cell viability was determined in the presence and absence of staurosporine (Fig. 4A). The addition of 10 nM staurosporine for 24 hours left 43% of RGCs viable (P < 0.001). The addition of 100 nM staurosporine for 24 hours left 41% of RGCs viable (P < 0.001). The addition of 1 μM staurosporine for 24 hours left 35% of RGCs viable (P < 0.001). The addition of 10 μM staurosporine for 24 hours left 0% of RGCs viable (P < 0.001). The 100 nM staurosporine concentration was used for all subsequent staurosporine/cell viability experiments (Fig. 4B). 
Figure 4. 
 
Staurosporine toxicity concentration-response. Staurosporine significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning staurosporine treatment. Cells were treated with or without varying concentrations of staurosporine (10 nM, 100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) Staurosporine significantly killed primary RGCs at 10 nM (43% survival P < 0.001), 100 nM (41% survival P < 0.001), 1 μM (35% survival P < 0.001), and 10 μM (0% survival P < 0.001) (n = 3).
Figure 4. 
 
Staurosporine toxicity concentration-response. Staurosporine significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning staurosporine treatment. Cells were treated with or without varying concentrations of staurosporine (10 nM, 100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) Staurosporine significantly killed primary RGCs at 10 nM (43% survival P < 0.001), 100 nM (41% survival P < 0.001), 1 μM (35% survival P < 0.001), and 10 μM (0% survival P < 0.001) (n = 3).
MB Protects Primary RGCs against Staurosporine
The addition of 100 nM staurosporine for 24 hours induced a significant level of RGC death (Fig. 5A). Untreated cells were used as a control to quantitate the average amount of viable cells following primary RGC isolation and will be set at 100% survival for the experiments (percentage of viable RGCs experimental group/percentage of viable RGCs vehicle group). The addition of 100 nM staurosporine in the presence of 100 nM MB did not induce a significant level of protection, whereas 1 μM and 10 μM MB significantly protected against 1 μM staurosporine toxicity (1.8-fold increase of RGC survival, P < 0.001, 1.9-fold increase RGC survival, P < 0.001) (Fig. 5B). Cells were not subjected to concentrations of MB over 10 μM due to toxic effects at higher doses. 31,32  
Figure 5. 
 
MB may protect primary RGCs from staurosporine. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning staurosporine treatment. Cells were treated with 100 nM staurosporine and with or without varying concentrations of MB (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). All other experimental groups will be expressed as a ratio of vehicle cell survival. (B) The addition of 100 nM staurosporine for 24 hours induced a significant level of RGC death. The addition of 100 nM staurosporine with 100 nM MB did not induce a significant level of protection. The addition of 1 μM and 10 μM MB significantly protected against 1 μM staurosporine toxicity (1.8-fold increase of RGC survival, P < 0.001, 1.9-fold increase of RGC survival, P < 0.001) (n = 6).
Figure 5. 
 
MB may protect primary RGCs from staurosporine. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning staurosporine treatment. Cells were treated with 100 nM staurosporine and with or without varying concentrations of MB (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). All other experimental groups will be expressed as a ratio of vehicle cell survival. (B) The addition of 100 nM staurosporine for 24 hours induced a significant level of RGC death. The addition of 100 nM staurosporine with 100 nM MB did not induce a significant level of protection. The addition of 1 μM and 10 μM MB significantly protected against 1 μM staurosporine toxicity (1.8-fold increase of RGC survival, P < 0.001, 1.9-fold increase of RGC survival, P < 0.001) (n = 6).
Hypoxia Time-Response
Tissue hypoxia in the optic nerve head and retina is thought to develop as secondary response following elevated IOP and is associated with the pathology underlying optic nerve degeneration. 33 Cells were incubated at 37°C in 0.5% O2, 10% CO2, and air for 24 to 72 hours (Fig. 6A). There was not a significant amount of RGC cell death at 24 hours following hypoxia (93% survival), whereas hypoxia significantly killed the primary RGCs at 48 hours, leaving 71% of the cells viable (P < 0.001). Hypoxia significantly killed the primary RGCs at 72 hours, leaving 62% of the cells viable (P < 0.001) (Fig. 6B). 
Figure 6. 
 
Hypoxia time-response. Hypoxia significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning hypoxia (0.5% oxygen). Cells were exposed to either hypoxic or normoxic conditions from 24 hours or 48 hours (scale bar = 200 μm). (B) There was not a significant amount of death at 24 hours of hypoxia (93% survival). Hypoxia significantly killed the primary RGCs at 48 hours, leaving 71% of the cells viable (P < 0.001). Hypoxia significantly killed the primary RGCs at 72 hours, leaving 62% of the cells viable (P < 0.001). Primary RGCs will be exposed to 72 hours of hypoxia for subsequential experiments (n = 6).
Figure 6. 
 
Hypoxia time-response. Hypoxia significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning hypoxia (0.5% oxygen). Cells were exposed to either hypoxic or normoxic conditions from 24 hours or 48 hours (scale bar = 200 μm). (B) There was not a significant amount of death at 24 hours of hypoxia (93% survival). Hypoxia significantly killed the primary RGCs at 48 hours, leaving 71% of the cells viable (P < 0.001). Hypoxia significantly killed the primary RGCs at 72 hours, leaving 62% of the cells viable (P < 0.001). Primary RGCs will be exposed to 72 hours of hypoxia for subsequential experiments (n = 6).
MB Protects Primary RGCs against Hypoxia
Cells were incubated at 37°C in 0.5% O2, 10% CO2, and air for 72 hours with or without MB. Hypoxia for 72 hours induced a significant level of RGC death. There was not a significant amount of protection after 72 hours with 100 nM MB treatment (Fig. 7A); however, MB, at concentrations of 1 μM and 10 μM, significantly protected primary RGCs from 72 hours of hypoxia (1.5-fold increase of RGC survival, P = 0.001, 1.7-fold increase of RGC survival, P < 0.001) (Fig. 7B). 
Figure 7. 
 
MB protects primary RGCs against hypoxia. Hypoxia significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning hypoxia (0.5% oxygen). Cells were exposed to either hypoxic or normoxic conditions from 24 hours or 48 hours (scale bar = 500 μm). (B) Seventy-two hours of hypoxic conditions induced a significant level of RGC death. There was not a significant amount of protection after 72 hours of 100 nM MB treatment. MB, at concentrations 1 μM and 10 μM, significantly protected primary RGCs from 72 hours of hypoxia (1.5-fold increase in RGC survival, P = 0.001, 1.7-fold increase of RGC survival, P < 0.001) (n = 6).
Figure 7. 
 
MB protects primary RGCs against hypoxia. Hypoxia significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning hypoxia (0.5% oxygen). Cells were exposed to either hypoxic or normoxic conditions from 24 hours or 48 hours (scale bar = 500 μm). (B) Seventy-two hours of hypoxic conditions induced a significant level of RGC death. There was not a significant amount of protection after 72 hours of 100 nM MB treatment. MB, at concentrations 1 μM and 10 μM, significantly protected primary RGCs from 72 hours of hypoxia (1.5-fold increase in RGC survival, P = 0.001, 1.7-fold increase of RGC survival, P < 0.001) (n = 6).
MB Increases Cytochrome c Oxidase Activity during Hydrogen Peroxide Toxicity
To determine if MB preserves activity of the electron transport chain in RGCs during a toxic challenge, we directly measure cytochrome c oxidase (Complex IV) activity. Approximately 400,000 RGCs were seeded in each well of a six-well plate. The cells were incubated for 7 days before being challenged with 500 μM hydrogen peroxide for 2 hours. Cytochrome c oxidase activity is expressed as a percentage to the control group. The cytochrome c oxidase activity of control RGCs was set to the arbitrary unit of 1, whereas every experimental group was expressed as a percentage of control. Hydrogen peroxide significantly decreased cytochrome c oxidase activity by 69% ± 7% of the control (P = 0.029). The addition of MB significantly preserved cytochrome c oxidase activity 91% ± 3 % (P = 0.037) against the hydrogen peroxide insult (Fig. 8). 
Figure 8. 
 
MB significantly increases cytochrome c oxidase activity during hydrogen peroxide toxicity. MB significantly protects cytochrome c activity in RGCs exposed to hydrogen peroxide; 400,000 primary rat RGCs were cultured for 7 days before being subjected to treatment. Primary RGCs were treated for 120 minutes with a vehicle or 500 μM hydrogen peroxide with or without 1 μM MB. Cytochrome c oxidase activity was measured by a cytochrome c oxidase assay kit (CYTOCOX1; Sigma-Aldrich) following the manufacturer's protocol. This assay uses cytochrome c oxidase activity to convert ferrocytochrome c, which has an absorbance at 550 nm, to ferricytochrome c, which does not have an absorbance at 550 nm. A spectrophotometer was used to measure ferrocytochrome at 550 nm for the course of 120 seconds. The faster the rate of change in absorbance at 550 nm indicates a larger amount of cytochrome c activity. Results indicate that primary RGCs had the highest functioning cytochrome c oxidase, whereas hydrogen peroxide decreased it. The addition of MB increased the amount of functioning cytochrome c oxidase. The control RGC cytochrome c oxidase activity was set to the arbitrary unit of 1, whereas every experimental group was expressed as a percentage of control. Hydrogen peroxide significantly decreased cytochrome c oxidase activity by 69% ± 7% of the control (P = 0.029). The addition of MB significantly preserved cytochrome c oxidase activity 91% ± 3 % (P = 0.037) against the hydrogen peroxide insult (n = 4).
Figure 8. 
 
MB significantly increases cytochrome c oxidase activity during hydrogen peroxide toxicity. MB significantly protects cytochrome c activity in RGCs exposed to hydrogen peroxide; 400,000 primary rat RGCs were cultured for 7 days before being subjected to treatment. Primary RGCs were treated for 120 minutes with a vehicle or 500 μM hydrogen peroxide with or without 1 μM MB. Cytochrome c oxidase activity was measured by a cytochrome c oxidase assay kit (CYTOCOX1; Sigma-Aldrich) following the manufacturer's protocol. This assay uses cytochrome c oxidase activity to convert ferrocytochrome c, which has an absorbance at 550 nm, to ferricytochrome c, which does not have an absorbance at 550 nm. A spectrophotometer was used to measure ferrocytochrome at 550 nm for the course of 120 seconds. The faster the rate of change in absorbance at 550 nm indicates a larger amount of cytochrome c activity. Results indicate that primary RGCs had the highest functioning cytochrome c oxidase, whereas hydrogen peroxide decreased it. The addition of MB increased the amount of functioning cytochrome c oxidase. The control RGC cytochrome c oxidase activity was set to the arbitrary unit of 1, whereas every experimental group was expressed as a percentage of control. Hydrogen peroxide significantly decreased cytochrome c oxidase activity by 69% ± 7% of the control (P = 0.029). The addition of MB significantly preserved cytochrome c oxidase activity 91% ± 3 % (P = 0.037) against the hydrogen peroxide insult (n = 4).
Discussion
Glaucoma is classified as a neurodegenerative disease characterized by the progressive loss of RGCs, leading to the loss of visual field and eventually blindness. Glaucoma is the second leading cause of blindness worldwide and the most common form of glaucoma, primary open angle glaucoma, accounts for 90% of the cases. 8 Although changes in the outflow pathway cause increased IOP, the actual mechanism responsible for optic nerve damage is still unclear. Mitochondrial dysfunction is believed to contribute to the pathogenesis observed during glaucoma. 1  
During the disease state of glaucoma, mitochondria are densely concentrated at the optic nerve head, which indicates the high recruitment of adenosine triphosphate (ATP) at the primary site of glaucomatous axonal injury. 34 When cultured RGCs are exposed to elevated hydrostatic pressure, the pressure induces mitochondrial fission and disruption of ATP production and predisposing the cells for apoptosis. 35,36 A mitochondrial enhancing compound, such as MB, could be used to protect against the mitochondrial impairment. MB is especially intriguing due to its low toxicity because it could be prescribed to groups of people who have a higher genetic prevalence to the disease. 37,38 MB is a potent metabolic-enhancing and antioxidant agent that facilitates memory and promotes neuroprotection. 17,24,25,39 MB can cycle between reduction and oxidation. These actions supports electron cycling of the electron transport chain. 40 This property allows MB to transfer electrons to oxygen, which is an essential process that exists in the electron transport chain of the mitochondria. 40 MB cannot enter a neuron until it is reduced (MBH2) at the cell surface. Once MBH2 enters the cell it can be re-oxidized back into MB, maintaining it within the cell. 41 MB may be re-oxidized by a heme-protein, such as cytochrome c or cytochrome c oxidase. 42 Within the cell, MB and MBH2 are maintained at equilibrium, making a reversible reduction-oxidation system. 43  
Our results indicated that MB preserved mitochondrial activity against oxidative stress caused by hydrogen peroxide–induced mitochondrial dysfunction, specifically involving cytochrome c oxidase. 44 This suggests that the neuroprotective agent, MB, may prevent mitochondrial dysfunction during glaucoma, thus leading to increased RGC survival and preserved vision; however, further testing is needed. 
MB significantly protected RGCs during three challenges that we examined. This includes protection against mitochondrial dysfunction induced by rotenone, which selectively impairs complex I of the electron transport chain. 30 MB is known to significantly protect against rotenone and other hermetic decreasing agents. 4,11,45 Our results indicate that MB's protection against rotenone is preserved within the RGCs of rats. 
Apoptosis is a common form of neuronal death during several neuronal diseases including glaucoma. 46,47 Even though the exact mechanism is unknown, staurosporine activates caspases and induces apoptosis. 48,49 Our results indicate that MB protects against staurosporine cytotoxicity. Even though the exact mechanism of this protection has not been thoroughly examined or is not completely understood, it suggests that MB may protect against apoptosis. 
Hypoxia and ischemia occur during age-related neurodegenerative diseases, including glaucoma and neurotraumas. 50,51 Hypoxia, oxygen deprivation, in neurons leads to a localized increase in excitatory amino acids and proteins that can result in the premature death of neurons and brain tissue. 52 MB increases neuronal survival during in vivo ischemia. 53,54 MB's mechanism of action suggests that it will increase the propagation of electrons, even during oxygen deprivation. Furthermore, MB is a free radical scavenger and can convert superoxide into water, 17 which can increase the viability of neurons during oxygen deprivation. Furthermore, our preliminary data suggest that MB's mechanism of action is preserved in RGCs by increasing the activity of cytochrome c oxidase (complex IV of the electron transport chain) 55 ; however, additional tests are needed. 
Although MB can protect retinal tissue during rotenone toxicity, 23,24 it would be beneficial to test if MB would be advantageous to develop as a treatment for the neurodegeneration that occurs during glaucoma. For this, a more sentient animal with more relevant models of glaucoma should be used. For example, examine if MB can protect RGC loss in primates following elevation of IOP. Such data would support the potential use of MB in the treatment of glaucoma. 
Acknowledgments
We sincerely thank Ben Barres for his time and help with the primary RGC isolation technique. Abe Clark is also greatly appreciated for his time and helpful discussions. 
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Footnotes
 Supported by Department of Defense Grant W81XWH-06-1-0290.
Footnotes
 Disclosure: D.R. Daudt III, None; B. Mueller, None; Y.H. Park, None; Y. Wen, None; T. Yorio, None
Figure 1. 
 
Primary rat RGC culture. Characterization of the isolated primary RGCs following Ben Barres' protocol. 29 (A) Cells were characterized by immunocytochemistry for normally expressed RGC marker Thy-1. (B) A blank of the same field was used. Thy-1 was detectable in virtually all of these cells. Scale bar, 100 μm.
Figure 1. 
 
Primary rat RGC culture. Characterization of the isolated primary RGCs following Ben Barres' protocol. 29 (A) Cells were characterized by immunocytochemistry for normally expressed RGC marker Thy-1. (B) A blank of the same field was used. Thy-1 was detectable in virtually all of these cells. Scale bar, 100 μm.
Figure 2. 
 
Rotenone toxicity concentration-response. Rotenone significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning rotenone treatment. Cells were treated with or without varying concentrations of rotenone (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) Rotenone significantly killed primary RGCs at 100 nM (59% survival P < 0.001), 1 μM (39% survival P < 0.001), and 10 μM (7% survival P < 0.001) (n = 3).
Figure 2. 
 
Rotenone toxicity concentration-response. Rotenone significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning rotenone treatment. Cells were treated with or without varying concentrations of rotenone (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) Rotenone significantly killed primary RGCs at 100 nM (59% survival P < 0.001), 1 μM (39% survival P < 0.001), and 10 μM (7% survival P < 0.001) (n = 3).
Figure 3. 
 
MB protects primary RGCs from rotenone toxicity. MB significantly protects primary RGCs from rotenone toxicity. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning rotenone treatment. Cells were treated with 1 μM rotenone and with or without varying concentrations of MB (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) A total of 1 μM rotenone significantly (41% survival P < 0.001) killed primary RGCs. MB significantly protected primary RGCs from rotenone toxicity at 1 μM (1.6-fold increase of RGC survival P < 0.001), and 10 μM (1.6-fold increase of RGC survival P < 0.001) (n = 6).
Figure 3. 
 
MB protects primary RGCs from rotenone toxicity. MB significantly protects primary RGCs from rotenone toxicity. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning rotenone treatment. Cells were treated with 1 μM rotenone and with or without varying concentrations of MB (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) A total of 1 μM rotenone significantly (41% survival P < 0.001) killed primary RGCs. MB significantly protected primary RGCs from rotenone toxicity at 1 μM (1.6-fold increase of RGC survival P < 0.001), and 10 μM (1.6-fold increase of RGC survival P < 0.001) (n = 6).
Figure 4. 
 
Staurosporine toxicity concentration-response. Staurosporine significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning staurosporine treatment. Cells were treated with or without varying concentrations of staurosporine (10 nM, 100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) Staurosporine significantly killed primary RGCs at 10 nM (43% survival P < 0.001), 100 nM (41% survival P < 0.001), 1 μM (35% survival P < 0.001), and 10 μM (0% survival P < 0.001) (n = 3).
Figure 4. 
 
Staurosporine toxicity concentration-response. Staurosporine significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning staurosporine treatment. Cells were treated with or without varying concentrations of staurosporine (10 nM, 100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). (B) Staurosporine significantly killed primary RGCs at 10 nM (43% survival P < 0.001), 100 nM (41% survival P < 0.001), 1 μM (35% survival P < 0.001), and 10 μM (0% survival P < 0.001) (n = 3).
Figure 5. 
 
MB may protect primary RGCs from staurosporine. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning staurosporine treatment. Cells were treated with 100 nM staurosporine and with or without varying concentrations of MB (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). All other experimental groups will be expressed as a ratio of vehicle cell survival. (B) The addition of 100 nM staurosporine for 24 hours induced a significant level of RGC death. The addition of 100 nM staurosporine with 100 nM MB did not induce a significant level of protection. The addition of 1 μM and 10 μM MB significantly protected against 1 μM staurosporine toxicity (1.8-fold increase of RGC survival, P < 0.001, 1.9-fold increase of RGC survival, P < 0.001) (n = 6).
Figure 5. 
 
MB may protect primary RGCs from staurosporine. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning staurosporine treatment. Cells were treated with 100 nM staurosporine and with or without varying concentrations of MB (100 nM, 1 μM, or 10 μM) for 24 hours (scale bar = 200 μm). All other experimental groups will be expressed as a ratio of vehicle cell survival. (B) The addition of 100 nM staurosporine for 24 hours induced a significant level of RGC death. The addition of 100 nM staurosporine with 100 nM MB did not induce a significant level of protection. The addition of 1 μM and 10 μM MB significantly protected against 1 μM staurosporine toxicity (1.8-fold increase of RGC survival, P < 0.001, 1.9-fold increase of RGC survival, P < 0.001) (n = 6).
Figure 6. 
 
Hypoxia time-response. Hypoxia significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning hypoxia (0.5% oxygen). Cells were exposed to either hypoxic or normoxic conditions from 24 hours or 48 hours (scale bar = 200 μm). (B) There was not a significant amount of death at 24 hours of hypoxia (93% survival). Hypoxia significantly killed the primary RGCs at 48 hours, leaving 71% of the cells viable (P < 0.001). Hypoxia significantly killed the primary RGCs at 72 hours, leaving 62% of the cells viable (P < 0.001). Primary RGCs will be exposed to 72 hours of hypoxia for subsequential experiments (n = 6).
Figure 6. 
 
Hypoxia time-response. Hypoxia significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning hypoxia (0.5% oxygen). Cells were exposed to either hypoxic or normoxic conditions from 24 hours or 48 hours (scale bar = 200 μm). (B) There was not a significant amount of death at 24 hours of hypoxia (93% survival). Hypoxia significantly killed the primary RGCs at 48 hours, leaving 71% of the cells viable (P < 0.001). Hypoxia significantly killed the primary RGCs at 72 hours, leaving 62% of the cells viable (P < 0.001). Primary RGCs will be exposed to 72 hours of hypoxia for subsequential experiments (n = 6).
Figure 7. 
 
MB protects primary RGCs against hypoxia. Hypoxia significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning hypoxia (0.5% oxygen). Cells were exposed to either hypoxic or normoxic conditions from 24 hours or 48 hours (scale bar = 500 μm). (B) Seventy-two hours of hypoxic conditions induced a significant level of RGC death. There was not a significant amount of protection after 72 hours of 100 nM MB treatment. MB, at concentrations 1 μM and 10 μM, significantly protected primary RGCs from 72 hours of hypoxia (1.5-fold increase in RGC survival, P = 0.001, 1.7-fold increase of RGC survival, P < 0.001) (n = 6).
Figure 7. 
 
MB protects primary RGCs against hypoxia. Hypoxia significantly kills primary rat RGCs. (A) Cell survival was measured by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. Primary rat RGCs were seeded, 25,000 per well. Cells were cultured for 72 hours before beginning hypoxia (0.5% oxygen). Cells were exposed to either hypoxic or normoxic conditions from 24 hours or 48 hours (scale bar = 500 μm). (B) Seventy-two hours of hypoxic conditions induced a significant level of RGC death. There was not a significant amount of protection after 72 hours of 100 nM MB treatment. MB, at concentrations 1 μM and 10 μM, significantly protected primary RGCs from 72 hours of hypoxia (1.5-fold increase in RGC survival, P = 0.001, 1.7-fold increase of RGC survival, P < 0.001) (n = 6).
Figure 8. 
 
MB significantly increases cytochrome c oxidase activity during hydrogen peroxide toxicity. MB significantly protects cytochrome c activity in RGCs exposed to hydrogen peroxide; 400,000 primary rat RGCs were cultured for 7 days before being subjected to treatment. Primary RGCs were treated for 120 minutes with a vehicle or 500 μM hydrogen peroxide with or without 1 μM MB. Cytochrome c oxidase activity was measured by a cytochrome c oxidase assay kit (CYTOCOX1; Sigma-Aldrich) following the manufacturer's protocol. This assay uses cytochrome c oxidase activity to convert ferrocytochrome c, which has an absorbance at 550 nm, to ferricytochrome c, which does not have an absorbance at 550 nm. A spectrophotometer was used to measure ferrocytochrome at 550 nm for the course of 120 seconds. The faster the rate of change in absorbance at 550 nm indicates a larger amount of cytochrome c activity. Results indicate that primary RGCs had the highest functioning cytochrome c oxidase, whereas hydrogen peroxide decreased it. The addition of MB increased the amount of functioning cytochrome c oxidase. The control RGC cytochrome c oxidase activity was set to the arbitrary unit of 1, whereas every experimental group was expressed as a percentage of control. Hydrogen peroxide significantly decreased cytochrome c oxidase activity by 69% ± 7% of the control (P = 0.029). The addition of MB significantly preserved cytochrome c oxidase activity 91% ± 3 % (P = 0.037) against the hydrogen peroxide insult (n = 4).
Figure 8. 
 
MB significantly increases cytochrome c oxidase activity during hydrogen peroxide toxicity. MB significantly protects cytochrome c activity in RGCs exposed to hydrogen peroxide; 400,000 primary rat RGCs were cultured for 7 days before being subjected to treatment. Primary RGCs were treated for 120 minutes with a vehicle or 500 μM hydrogen peroxide with or without 1 μM MB. Cytochrome c oxidase activity was measured by a cytochrome c oxidase assay kit (CYTOCOX1; Sigma-Aldrich) following the manufacturer's protocol. This assay uses cytochrome c oxidase activity to convert ferrocytochrome c, which has an absorbance at 550 nm, to ferricytochrome c, which does not have an absorbance at 550 nm. A spectrophotometer was used to measure ferrocytochrome at 550 nm for the course of 120 seconds. The faster the rate of change in absorbance at 550 nm indicates a larger amount of cytochrome c activity. Results indicate that primary RGCs had the highest functioning cytochrome c oxidase, whereas hydrogen peroxide decreased it. The addition of MB increased the amount of functioning cytochrome c oxidase. The control RGC cytochrome c oxidase activity was set to the arbitrary unit of 1, whereas every experimental group was expressed as a percentage of control. Hydrogen peroxide significantly decreased cytochrome c oxidase activity by 69% ± 7% of the control (P = 0.029). The addition of MB significantly preserved cytochrome c oxidase activity 91% ± 3 % (P = 0.037) against the hydrogen peroxide insult (n = 4).
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