Photobiomodulation Directly Benefits Primary Neurons Functionally Inactivated by Toxins
Far red and near infrared (NIR) light promotes wound healing, but the mechanism is poorly understood. Our previous studies using 670 nm light-emitting diode (LED) arrays suggest that cytochrome c oxidase, a photoacceptor in the NIR range, plays an important role in therapeutic photobiomodulation. If this is true, then an irreversible inhibitor of cytochrome c oxidase, potassium cyanide (KCN), should compete with LED and reduce its beneficial effects. This hypothesis was tested on primary cultured neurons. LED treatment partially restored enzyme activity blocked by 10–100 μm KCN. It significantly reduced neuronal cell death induced by 300 μm KCN from 83.6 to 43.5%. However, at 1–100 mm KCN, the protective effects of LED decreased, and neuronal deaths increased. LED significantly restored neuronal ATP content only at 10 μm KCN but not at higher concentrations of KCN tested. Pretreatment with LED enhanced efficacy of LED during exposure to 10 or 100 μm KCN but did not restore enzyme activity to control levels. In contrast, LED was able to completely reverse the detrimental effect of tetrodotoxin, which only indirectly down-regulated enzyme levels. Among the wavelengths tested (670, 728, 770, 830, and 880 nm), the most effective ones (830 nm, 670 nm) paralleled the NIR absorption spectrum of oxidized cytochrome c oxidase, whereas the least effective wavelength, 728 nm, did not. The results are consistent with our hypothesis that the mechanism of photobiomodulation involves the up-regulation of cytochrome c oxidase, leading to increased energy metabolism in neurons functionally inactivated by toxins. Far red and near infrared (NIR) light promotes wound healing, but the mechanism is poorly understood. Our previous studies using 670 nm light-emitting diode (LED) arrays suggest that cytochrome c oxidase, a photoacceptor in the NIR range, plays an important role in therapeutic photobiomodulation. If this is true, then an irreversible inhibitor of cytochrome c oxidase, potassium cyanide (KCN), should compete with LED and reduce its beneficial effects. This hypothesis was tested on primary cultured neurons. LED treatment partially restored enzyme activity blocked by 10–100 μm KCN. It significantly reduced neuronal cell death induced by 300 μm KCN from 83.6 to 43.5%. However, at 1–100 mm KCN, the protective effects of LED decreased, and neuronal deaths increased. LED significantly restored neuronal ATP content only at 10 μm KCN but not at higher concentrations of KCN tested. Pretreatment with LED enhanced efficacy of LED during exposure to 10 or 100 μm KCN but did not restore enzyme activity to control levels. In contrast, LED was able to completely reverse the detrimental effect of tetrodotoxin, which only indirectly down-regulated enzyme levels. Among the wavelengths tested (670, 728, 770, 830, and 880 nm), the most effective ones (830 nm, 670 nm) paralleled the NIR absorption spectrum of oxidized cytochrome c oxidase, whereas the least effective wavelength, 728 nm, did not. The results are consistent with our hypothesis that the mechanism of photobiomodulation involves the up-regulation of cytochrome c oxidase, leading to increased energy metabolism in neurons functionally inactivated by toxins. Near infrared (NIR) 1The abbreviations used are: NIR, near infrared; LED, light-emitting diode; TTX, tetrodotoxin; KCN, potassium cyanide; NaN3, sodium azide.1The abbreviations used are: NIR, near infrared; LED, light-emitting diode; TTX, tetrodotoxin; KCN, potassium cyanide; NaN3, sodium azide. light has been used in therapeutic devices for the treatment of a variety of injuries, especially infected, ischemic, and hypoxic wounds (1Conlan M.J. Rapley J.W. Cobb C.M. J. Clin. Periodontol. 1996; 23: 492-496Crossref PubMed Scopus (385) Google Scholar, 2Sommer A.P. Pinheiro A.L. Mester A.R. Franke R.P. Whelan H.T. J. Clin. Laser Med. Surg. 2001; 19: 29-33Crossref PubMed Scopus (239) Google Scholar, 3Whelan H.T. Smits Jr., R.L. Buchman E.V. Whelan N.T. Turner S.G. Margolis D.A. Cevenini V. Stinson H. Ignatius R. Martin T. Cwiklinski J. Philippi A.F. Graf W.R. Hodgson B. Gould L. Kane M. Chen G. Caviness J. J. Clin. Laser Med. Surg. 2001; 19: 305-314Crossref PubMed Scopus (391) Google Scholar, 4Yu W. Naim J.O. Lanzafame R.J. Lasers Surg. Med. 1997; 20: 56-63Crossref PubMed Scopus (230) Google Scholar). NIR light penetrates more deeply than UV or visible light and is benign to living tissue. This presents clear clinical advantages to treatment within a tissue transparency window of 650–1000 nm. Most of the devices utilize lasers as the light source. Recently, however, light-emitting diodes (LEDs) have been found to be more beneficial than lasers in several respects (3Whelan H.T. Smits Jr., R.L. Buchman E.V. Whelan N.T. Turner S.G. Margolis D.A. Cevenini V. Stinson H. Ignatius R. Martin T. Cwiklinski J. Philippi A.F. Graf W.R. Hodgson B. Gould L. Kane M. Chen G. Caviness J. J. Clin. Laser Med. Surg. 2001; 19: 305-314Crossref PubMed Scopus (391) Google Scholar, 5Whelan H.T. Connelly J.F. Hodgson B.D. Barbeau L. Post A.C. Bullard G. Buchmann E.V. Kane M. Whelan N.T. Warwick A. Margolis D. J. Clin. Laser Med. Surg. 2002; 20: 319-324Crossref PubMed Scopus (123) Google Scholar). LEDs can be constructed to form relatively large arrays to treat wounds that commonly involve areas much larger than the size of a laser beam. Unlike lasers, there is virtually no heat generated by the LED array and therefore no potential thermal injury to individuals being treated. LED is well tolerated by biological tissues and has no known detrimental effect. As a therapeutic device, LED has achieved FDA non-significant risk status. Moreover, LED units are more compact, portable, and affordable than lasers. The cellular mechanisms of action of NIR in wound healing are not well understood. The basic premise is that long wavelength lights stimulate cellular energy metabolism and energy production. Three major photoacceptor molecules in mammalian tissues are known to absorb light in the NIR range: hemoglobin, myoglobin, and cytochrome c oxidase. Of the three, only cytochrome c oxidase (EC 1.9.3.1) has been associated with energy production. In our recent study, the first two candidates were excluded in our primary neuronal cultures in which more than 95% of the cells were neurons, and there were no blood elements or muscle cells (6Wong-Riley M.T. Bai X. Buchmann E. Whelan H.T. Neuroreport. 2001; 12: 3033-3037Crossref PubMed Scopus (127) Google Scholar). In the presence of a voltage-dependent sodium channel blocker, tetrodotoxin (TTX), which impedes neuronal impulse activity, decreases ATP demand, and down-regulates cytochrome c oxidase activity, LED at 670 nm was able to reverse the detrimental effect of TTX by bringing cytochrome c oxidase back to control levels. Moreover, LED treatment up-regulated enzyme activity of normal neurons above control levels (6Wong-Riley M.T. Bai X. Buchmann E. Whelan H.T. Neuroreport. 2001; 12: 3033-3037Crossref PubMed Scopus (127) Google Scholar). The beneficial effect of NIR was further confirmed in our recent in vivo study. Rats intoxicated with methanol developed retinal dysfunction attributable to the inhibition of cytochrome c oxidase by formic acid, the toxic metabolite in methanol intoxication. Three brief LED treatments significantly improved retinal function as measured by the electroretinographic response and protected the retina from histopathological changes induced by methanol-derived formate (7Eells J.T. M.T. Buchmann E.V. Kane M. Whelan N.T. Whelan H.T. A. PubMed Scopus Google Scholar). photobiomodulation is therapeutic in inhibition of cytochrome c oxidase by Our in and in vivo studies suggest that cytochrome c oxidase plays an important role in the therapeutic of photobiomodulation. of the action spectrum for cellular laser with the absorption of potential T. J. PubMed Scopus Google to suggest that cytochrome c oxidase is a primary photoacceptor of light in the red to near infrared that of near infrared light is by as cytochrome c oxidase B. T. B. J. PubMed Scopus Google Scholar). further this the tested the cytochrome c oxidase is in the photobiomodulation a and irreversible inhibitor of cytochrome c oxidase as potassium cyanide compete with and the beneficial effect of LED treatment cytochrome c oxidase activity in LED further the protective action of LED during exposure to KCN. cytochrome c oxidase activity and cellular LED reduce neuronal cell death by the KCN. the effective action spectrum of LED in the detrimental effect of the impulse tetrodotoxin on cytochrome c oxidase activity should to the NIR absorption spectrum of cytochrome c oxidase. wavelengths of LED were tested to ones were more beneficial to functionally inactivated neurons. were or in to the for neuronal cultures were our primary neurons are more than cell neurons are and can be to neurons from cell LED arrays 10 with wavelengths at 728, 770, 830, or 880 nm were from ATP cell cytochrome ATP and tetrodotoxin were from cyanide was from were or The for neurons from was as G. G. The Scholar, M. J. PubMed Scopus Google Scholar). a from was cultured in this was neurons from were cultured on with was of neurons to the of were with cells but were from by cultures at of were to 10 100 10 or 100 mm KCN for cell studies were in to of As a to the KCN of cultures were to 10 100 and mm of sodium for TTX, a of voltage-dependent sodium was to cultures at of at a of and cultures were in for TTX was a no light diode arrays of wavelengths were 728, 770, 830, and 880 nm, with a of nm at The was and energy was for and (6Wong-Riley M.T. Bai X. Buchmann E. Whelan H.T. Neuroreport. 2001; 12: 3033-3037Crossref PubMed Scopus (127) Google Scholar). KCN of to of KCN were with LED at 670 nm and energy of a on of the of exposure to KCN. of normal KCN of KCN but no Pretreatment of cultures were with LED at 670 nm and energy of and a for or being to 10 or 100 μm of KCN for during which LED treatment as were normal with LED for by 10 μm of KCN LED, 10 μm of KCN LED with LED for by 100 μm of KCN LED, and 100 μm of KCN LED to were the as the that and of of to TTX for were with of the wavelengths (670, 728, 770, 830, and 880 nm) at a of and energy of for and for the of the in the presence of of normal TTX of TTX but no the of or of treatments cytochrome c oxidase activity in neurons as with wavelength of cultures were to TTX for and were with LED of of wavelengths as 670 nm 728 nm 670 nm nm 728 728 nm 670 nm 728 nm nm 670 nm 670 nm 728 nm 728 nm 670 nm. wavelength a of and energy of was to a of wavelengths as a for the of the of exposure to 670 nm 728 nm nm 670 nm 728 nm nm 670 nm 728 nm nm 670 nm 728 nm 880 nm 670 nm 728 nm 880 nm 670 nm 728 nm 880 nm and were with a of wavelengths at the of which was for a of to an energy of treatments were in on the which was TTX and cells were of the cultures were not to TTX or to LED c oxidase were for control and cultures in an and cytochrome c in (6Wong-Riley M.T. Bai X. Buchmann E. Whelan H.T. Neuroreport. 2001; 12: 3033-3037Crossref PubMed Scopus (127) Google Scholar, M. J. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). cultures from the for the were for ATP content by of the using a of the of PubMed Google and of the cell cultured cells were with then in cell ATP for and from the by of a cell were then with the ATP and with a were as ATP content of The of normal to neurons cell death were with in cells to the of G. B. M. 1997; PubMed Scopus Google Scholar). were The of neurons with cell was from the of cells in of normal 100 μm KCN or 300 μm KCN exposure for and with 670 nm LED and for 10 exposure to of 100 μm or 300 μm KCN inhibitor of cytochrome c oxidase, NaN3, was tested at concentrations of 100 300 and mm and with the of changes in cytochrome c oxidase activity KCN or TTX exposure and LED of in cells from of the above were measured by of a to a two size were from the of and 300 cells were measured from of using a The was by a in and and were for and of for was used to and and of with within were by the were as of or was KCN (6Wong-Riley M.T. Bai X. Buchmann E. Whelan H.T. Neuroreport. 2001; 12: 3033-3037Crossref PubMed Scopus (127) Google Scholar, M. J. PubMed Scopus Google cultured neurons were and for cytochrome c oxidase. The was to to and KCN the activity of cytochrome c oxidase was reduced in and in and neurons As in and and KCN treatment in a in cytochrome c oxidase activity of primary neurons at the 10 μm concentrations of KCN from 10 μm to 100 mm in enzyme activity and neuronal neurons in were and for cytochrome c oxidase normal neurons to 10 μm KCN for neurons were with 670 nm LED being to 10 μm of KCN for neurons with 670 nm LED for being to 10 μm KCN for during which LED treatment was LED treatment at 670 nm of for and a for of the of exposure to concentrations of KCN significantly the detrimental effect of the on cytochrome c oxidase activity for neuronal However, were only for the neurons but than for the and neurons to as with and higher concentrations of KCN neuronal death and from the LED were for cell of 100 mm KCN were not most of the cells did not at that cultures to at concentrations 100 and in of cytochrome c oxidase activity not In to KCN, a higher of was to the of LED treatment partially the inhibition but not to control levels not to the with KCN. ATP concentrations to of cytochrome c oxidase in response to KCN or LED However, LED treatment was able to reverse the effect of only the of KCN tested but not at concentrations of 100 μm or The ATP content in cells to 10 mm KCN was the of the ATP and LED treatment or no effect. cells at 100 mm KCN for of ATP Pretreatment with LED at 670 nm of to and a for for neurons that LED treatment a during exposure to 10 or 100 μm of KCN and It significantly improved the of cytochrome c oxidase activity in and cell above by LED treatment during KCN exposure to However, the were significantly of normal for Pretreatment did not to have for the concentrations of KCN were not tested. Pretreatment with the a for further increased cytochrome c oxidase activity in neurons that LED during exposure to 10 μm or 100 μm of KCN to However, were significantly than of normal for of the cell to did not further for the cell that LED during exposure to 100 μm KCN. of of cell death in cultures and the effect of LED were with of cells whereas neurons cell death and with As in control cultures of cell This at 100 μm KCN and was not from that of with 10 of LED not concentrations of KCN in of neurons with to 300 μm KCN for a of neurons with 10 of LED at 670 nm to of energy reduced the of neurons with to of KCN with at mm generated of neurons with and with LED for 10 significantly reduced the of neurons cell death to the of cell death with 300 μm KCN and in cell death with LED not the wavelength for the detrimental effect of TTX on neuronal cytochrome c oxidase activity, wavelengths were 728, 770, 830, and 880 nm. was at a of and energy of to and for the of the of TTX As in 670 and nm were effective in the effect of TTX on cytochrome c oxidase activity, with nm being the most beneficial in completely enzyme activity to above not control levels 670 nm was effective in bringing nm was as beneficial as 880 nm the cell of enzyme whereas 880 nm for cell 728 nm, on the was the least effective and only of enzyme activity in neurons. The ATP content of neurons was reduced to of control levels by of exposure to μm of TTX LED treatments for 728 nm were able to completely reverse the detrimental nm to be effective in ATP content to above not significantly control levels 670 nm restored the ATP content to a above control levels and 880 nm were effective in bringing ATP content to and of control the 728 nm was the least effective and in the detrimental effect of TTX only of to the of TTX of wavelengths be more beneficial than tested of a for the of in TTX, at the energy of for that tested in cytochrome c oxidase levels above of for ATP content the of tested with at or above control levels The two of neurons a of LED on the of a TTX exposure and were was with LEDs at 728, and nm at for the first and at 10 for the and was to that nm was by 880 nm LED The energy was at for treatment to the a that completely the detrimental effect of TTX on cytochrome c oxidase activity, with the cell above control levels The cellular ATP content the with control levels c action of cytochrome c oxidase activity and ATP content of neurons to wavelengths were absorption spectrum of the enzyme R. 1997; PubMed Scopus Google Scholar). As in the effective especially 670 and nm, with the known absorption spectrum of oxidized cytochrome c oxidase. the the least effective wavelength, 728 nm, did not to the absorption spectrum of the of and with c the of cyanide and with cytochrome c oxidase with or LED The cyanide were for neurons as in and the were for cell from our The cyanide concentrations were 10 100 and 10 whereas for were 10 100 and In the of the were two to higher than the of B. PubMed Scopus Google Scholar). LED treatment reduced the of the for KCN and to for KCN and from to for The of the are consistent with our that cytochrome c oxidase is in the photobiomodulation of LED, the beneficial of LED are by KCN, a known inhibitor of the oxidase, LED exposure to KCN the effect of LED during KCN the action of effective wavelengths of LED to the absorption spectrum of oxidized cytochrome c oxidase in the near infrared range, and LED significantly the of cell death induced by KCN. KCN and TTX significantly down-regulated cytochrome c oxidase activity in neurons, and LED the detrimental effects of the toxins. However, the of for the two toxins. cyanide is a and irreversible inhibitor of cytochrome c oxidase concentrations of KCN, LED up-regulated the enzyme the action of KCN, but the control levels. higher concentrations of KCN, LED no enzyme activity as by cell LED and KCN have on the the TTX indirectly cytochrome c oxidase activity by neuronal action energy of functionally LED was able to completely reverse the detrimental effect of TTX by the enzyme to its control levels. found that a of is to a in cytochrome c oxidase activity of which of normal enzyme activity in neuronal M.T. PubMed Scopus Google Scholar). The is on in neurons, and the neuronal have levels of enzyme the of cytochrome c oxidase activity by 10 μm KCN in neurons was whereas that by μm TTX, a that impulse activity in neurons M.T. 12: PubMed Scopus Google or of the normal for neurons. that the activity of this enzyme is at its M.T. J. PubMed Google Scholar). the up-regulation of enzyme activity by LED is to involve of In the presence of TTX, LED is able to stimulate of of to the oxidase to its control In the of KCN however, the of be partially blocked by of KCN and completely blocked by of KCN. LED is from the enzyme to its control levels in the presence of KCN. The cellular content of ATP was reduced by KCN, and LED in the presence of 10 μm KCN was able to restore the ATP content to control levels. However, 100 μm or more of KCN the beneficial effect of LED in ATP that cytochrome c oxidase activity was partially enhanced by LED, was not for a of ATP only 10 of LED at a energy of reduced by the of neurons cell death in 300 μm KCN. LED significantly reduced the of cells with induced by inhibitor of cytochrome c oxidase, sodium azide. The of the is to be the of L. J. 2002; PubMed Scopus Google Scholar). The to cell death that within the tested LED is able to cytochrome c oxidase and ATP and to a that neurons from cell The of wavelengths the of 670 and nm were effective in cytochrome c oxidase to control levels in the presence of TTX, whereas 880 and nm were and 728 nm was the least results are to of T. J. PubMed Scopus Google in which the action spectrum of cell was treatment with lasers of In that study, found action in the red to near infrared range: and nm, which to absorption of of cytochrome c oxidase, reduced oxidized reduced and oxidized Our LEDs have an effective of nm, our 670 and nm treatments within the absorption of oxidized and oxidized nm is known to be the absorption spectrum of oxidized cytochrome c oxidase of the of A. PubMed Scopus Google Scholar, M. D.A. R.J. Med. PubMed Scopus Google Scholar, M. M. and is the most effective wavelength in the study. 880 nm within the of oxidized and nm is to the absorption spectrum of reduced whereas the spectrum of reduced nm) was not tested in the study. nm is to nm, the absorption of in cytochrome c oxidase of PubMed Scopus Google Scholar). to for nm is the known absorption for at nm B. H. R. M. W. M. H. R. A. PubMed Scopus Google Scholar). However, in the study, there was no blood that to the nm cytochrome c oxidase is the most the 728 nm was the least effective in our study, and that lasers within this did not The effective wavelengths with the known absorption spectrum of oxidized cytochrome c oxidase T. M. T. M. B. of the of The of Scholar). the of wavelengths and nm, M. V. M. E. R. J. L. J. Med. 1997; PubMed Scopus Google that most of the in vivo can be to cytochrome c oxidase. The wavelength efficacy and cytochrome c oxidase absorption spectrum our hypothesis that cytochrome c oxidase plays an important role in therapeutic photobiomodulation by LED H.T. Connelly J.F. Hodgson B.D. Barbeau L. Post A.C. Bullard G. Buchmann E.V. Kane M. Whelan N.T. Warwick A. Margolis D. J. Clin. Laser Med. Surg. 2002; 20: 319-324Crossref PubMed Scopus (123) Google Scholar, M.T. Bai X. Buchmann E. Whelan H.T. Neuroreport. 2001; 12: 3033-3037Crossref PubMed Scopus (127) Google Scholar, J.T. M.T. Buchmann E.V. Kane M. Whelan N.T. Whelan H.T. A. PubMed Scopus Google Scholar). As the in enzyme activity is to be a of an in the of the enzyme M.T. J. PubMed Google of LED treatment did not a from the control not whereas the a in activity was in the neurons (6Wong-Riley M.T. Bai X. Buchmann E. Whelan H.T. Neuroreport. 2001; 12: 3033-3037Crossref PubMed Scopus (127) Google Scholar). from our ATP the in the enzyme studies in that 670 and nm were able to restore ATP content to or above control levels in the presence of and 880 nm were to a whereas 728 nm was the least LED most the activity and of cytochrome c oxidase, in increased energy in neurons. treatments for wavelength tested results in cytochrome c oxidase activity above control levels. 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This is not there are of in however, and in our study, as there were no blood elements or muscle in our as cytochrome cytochrome and as well as be at this The that LED treatment a in the of the for cyanide and that LED induced an in the of cytochrome c oxidase consistent with our involves a that or indirectly and of cytochrome c oxidase from the and The cellular effect of brief LED treatments further that there is an of a of leading to the and of a variety of Whelan H. A. R. R. J. M. 2002; Scholar, H.T. Buchmann E.V. A. Kane Whelan N.T. M.T. J.T. Gould R. R. M. J. Clin. Laser Med. Surg. PubMed Scopus Google Scholar). PubMed Scopus Google have that to with cytochrome c oxidase be by changes in the by the of or as well as by the and the molecules that light absorption with as H. PubMed Scopus Google and and A.R. D. PubMed Scopus Google further In photoacceptor molecules to be the our hypothesis that the cytochrome c oxidase, is an important biological photoacceptor that photobiomodulation in the red and near infrared It further that the ATP content of primary neurons or in with the activity of cytochrome c oxidase, a known The therapeutic effects of photobiomodulation in wound healing (1Conlan M.J. Rapley J.W. Cobb C.M. J. Clin. Periodontol. 1996; 23: 492-496Crossref PubMed Scopus (385) Google Scholar, 2Sommer A.P. Pinheiro A.L. Mester A.R. Franke R.P. Whelan H.T. J. Clin. Laser Med. Surg. 2001; 19: 29-33Crossref PubMed Scopus (239) Google Scholar, 3Whelan H.T. Smits Jr., R.L. Buchman E.V. Whelan N.T. Turner S.G. Margolis D.A. Cevenini V. Stinson H. Ignatius R. Martin T. Cwiklinski J. Philippi A.F. Graf W.R. Hodgson B. Gould L. Kane M. Chen G. Caviness J. J. Clin. Laser Med. Surg. 2001; 19: 305-314Crossref PubMed Scopus (391) Google Scholar, 4Yu W. Naim J.O. Lanzafame R.J. Lasers Surg. Med. 1997; 20: 56-63Crossref PubMed Scopus (230) Google Scholar, 5Whelan H.T. Connelly J.F. Hodgson B.D. Barbeau L. Post A.C. Bullard G. Buchmann E.V. Kane M. Whelan N.T. Warwick A. Margolis D. J. Clin. Laser Med. Surg. 2002; 20: 319-324Crossref PubMed Scopus (123) Google and in by that cytochrome c oxidase in M.T. Bai X. Buchmann E. Whelan H.T. Neuroreport. 2001; 12: 3033-3037Crossref PubMed Scopus (127) Google Scholar, and in vivo (7Eells J.T. M.T. Buchmann E.V. Kane M. Whelan N.T. Whelan H.T. A. PubMed Scopus Google are are clear clinical advantages to treatment using wavelengths within the tissue transparency window of 650–1000 nm. of this especially with the risk LED can and should be for biological and as that involve cellular metabolism and energy production. and for and the of in the light-emitting diode arrays for this study.