Methylene blue protects primary rat retinal ganglion cells from cellular senescence

Donald R. Daudt, Brett Mueller, Yong H. Park, Yi Wen, Thomas Yorio

Research output: Contribution to journalArticleResearchpeer-review

22 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)4657-4667
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume53
Issue number8
DOIs
StatePublished - 1 Jul 2012

Fingerprint

Retinal Ganglion Cells
Cell Aging
Methylene Blue
Electron Transport Complex IV
Rotenone
Optic Nerve Diseases
Staurosporine
Electron Transport
Ubiquinone
Poisons
Neuroprotective Agents
Optic Nerve
Cytochromes c
Glaucoma
NAD
Hydrogen Peroxide
Sprague Dawley Rats
Cell Survival
Mitochondria

Cite this

Daudt, Donald R. ; Mueller, Brett ; Park, Yong H. ; Wen, Yi ; Yorio, Thomas. / Methylene blue protects primary rat retinal ganglion cells from cellular senescence. In: Investigative Ophthalmology and Visual Science. 2012 ; Vol. 53, No. 8. pp. 4657-4667.
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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.",
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Methylene blue protects primary rat retinal ganglion cells from cellular senescence. / Daudt, Donald R.; Mueller, Brett; Park, Yong H.; Wen, Yi; Yorio, Thomas.

In: Investigative Ophthalmology and Visual Science, Vol. 53, No. 8, 01.07.2012, p. 4657-4667.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Methylene blue protects primary rat retinal ganglion cells from cellular senescence

AU - Daudt, Donald R.

AU - Mueller, Brett

AU - Park, Yong H.

AU - Wen, Yi

AU - Yorio, Thomas

PY - 2012/7/1

Y1 - 2012/7/1

N2 - 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.

AB - 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.

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