Methylene blue protects primary rat retinal ganglion cells from cellular senescence.
OBJECTIVE
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.
Department of Pharmacology & Neuroscience, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA.
Donald R
DR
Daudt
Brett
B
Mueller
Yong H
YH
Park
Yi
Y
Wen
Thomas
T
Yorio
D000818:Animals; D016923:Cell Death / Q000187:drug effects*; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects / Q000502:physiology; D003576:Electron Transport Complex IV / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007150:Immunohistochemistry; D008751:Methylene Blue / Q000494:pharmacology*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D004791:Enzyme Inhibitors; D018696:Neuroprotective Agents; D014475:Uncoupling Agents; D012402:Rotenone; D003576:Electron Transport Complex IV; D019311:Staurosporine; D008751:Methylene Blue