Cytochrome C Oxidase

Sci Rep. 2016; 6: 30540.
Published online 2016 Aug 3. doi:  10.1038/srep30540
PMCID: PMC4971496

Interplay between up-regulation of cytochrome-c-oxidase and hemoglobin oxygenation induced by near-infrared laser

Xinlong Wang,1,* Fenghua Tian,1,* Sagar S. Soni,1 F. Gonzalez-Lima,a,2 and Hanli Liub,1
1Department of Bioengineering, the University of Texas at Arlington, 500 UTA Blvd, Arlington, TX 76010, USA
2Department of Psychology and Institute for Neuroscience, the University of Texas at Austin, 108 E. Dean Keeton Stop A8000, Austin, TX 78712, USA.
*These authors contributed equally to this work.
Author information ? Article notes ? Copyright and License information ?
Received 2016 Apr 14; Accepted 2016 Jul 6.

Abstract

Photobiomodulation, also known as low-level laser/light therapy (LLLT), refers to the use of red-to-near-infrared light to stimulate cellular functions for physiological or clinical benefits. The mechanism of LLLT is assumed to rely on photon absorption by cytochrome c oxidase (CCO), the terminal enzyme in the mitochondrial respiratory chain that catalyzes the reduction of oxygen for energy metabolism. In this study, we used broadband near-infrared spectroscopy (NIRS) to measure the LLLT-induced changes in CCO and hemoglobin concentrations in human forearms in vivo. Eleven healthy participants were administered with 1064-nm laser and placebo treatments on their right forearms. The spectroscopic data were analyzed and fitted with wavelength-dependent, modified Beer-Lambert Law. We found that LLLT induced significant increases of CCO concentration (?[CCO]) and oxygenated hemoglobin concentration (?[HbO]) on the treated site as the laser energy dose accumulated over time. A strong linear interplay between ?[CCO] and ?[HbO] was observed for the first time during LLLT, indicating a hemodynamic response of oxygen supply and blood volume closely coupled to the up-regulation of CCO induced by photobiomodulation. These results demonstrate the tremendous potential of broadband NIRS as a non-invasive, in vivo means to study mechanisms of photobiomodulation and perform treatment evaluations of LLLT.

Low-level laser/light therapy (LLLT), also known as photobiomodulation, refers to the use of low-level light in the red-to-near-infrared range (620–1100?nm) to stimulate cellular functions for physiological or clinical benefits. Photobiomodulation has been used to improve wound healing1,2, reduce pain3,4, and many other human applications. The light can be supplied by lasers or light-emitting diodes (LEDs). In recent years, transcranial LLLT has gained increased recognition for its therapeutic use in various neurological and psychological conditions, including ischemic stroke5,6, chronic traumatic brain injuries7,8, and depression9,10. Furthermore, using a 1064-nm laser, Barrett and Gonzalez-Lima conducted the first placebo-controlled studies demonstrating that LLLT to the forehead benefits cognition in healthy humans, including enhanced attention, working memory, and executive functions11,12,13.

The mechanism of photobiomodulation is proposed to rest on photon absorption by cytochrome c oxidase (CCO)14, the terminal enzyme in the mitochondrial respiratory chain that catalyzes the reduction of oxygen for energy metabolism1,2,15,16. The more the activity of CCO increases, the more oxygen consumption and metabolic energy is produced via mitochondrial oxidative phosphorylation17. Since CCO is an inducible enzyme, a longer-lasting metabolic effect is achieved by LLLT’s up-regulating CCO concentration, which in turn enhances the capacity for cellular oxygen metabolism [13]. Because neurons are cells highly dependent on oxygen metabolism, this photonics-bioenergetics mechanism results in metabolic and hemodynamic alterations that facilitate neuronal functioning15,18. To date, most research into effects of LLLT on mitochondrial enzymes has been conducted in cultured neurons1,2 and animal brains19 with invasive means. There is lack of experimental or direct observation on how LLLT modulates CCO levels and how the up-regulated enzyme affects or interplays with hemodynamic oxygenation in human tissues in vivo. The primary goal of this study was to utilize an experimental optical imaging approach to observe LLLT-induced up-regulation of CCO and its relationship with hemoglobin oxygenation in human forearms for better understanding and validation of photobiomodulation in vivo effects.

Near-infrared spectroscopy (NIRS)20 is a non-invasive and portable technology that can be used to probe biological and physiological states of living tissues based on the level of absorption and scattering of near-infrared light. In the past two decades, NIRS has been broadly investigated for quantification of oxygenated and deoxygenated hemoglobin concentrations (i.e., [HbO] and [Hb], respectively) in a variety of tissues21, such as the human breast22,23,24, the human prostate25,26, and the human brain24,27, in order to diagnose cancers or to map/image functional brain activities in vivo. In conventional NIRS, two or three wavelengths are adequately employed for characterizing cerebral or tissue HbO and Hb concentrations under different stimulations or psychological conditions. Based on the same working principle, we have recently utilized dual-wavelength NIRS to assess the hemodynamic effects of transcranial LLLT in the human brain in vivo. We found that transcranial 1064-nm laser improved cerebral oxygenation, indicated by an increase of [HbO] and a decrease of [Hb] in a dose-dependent manner28. However, because of the limited number of measuring wavelengths in dual-wavelength NIRS, we were not able to reliably quantify any change or elevation of CCO. As the mechanistic action of LLLT relies on direct photoactivation of CCO, it is crucial to quantify the LLLT-induced CCO changes as well. Thus, further improvement in our methodology was sought in order to address this critical need.

Since the initial development of NIRS technology, a significant amount of research effort has been persistently made to utilize broadband NIRS (bb-NIRS) for calculating the redox state of CCO based on its absorption band at 820–840?nm29. While the actual implementation of this approach started more than 20 years ago30,31, it had uncertainty on the accuracy of the methodology29,32,33. It is only in recent years when bb-NIRS has been reported by numerous publications to be a reliable means for computing both cerebral hemoglobin and CCO concentration changes during brain activations34,35,36 and/or brain injury37,38. Therefore, bb-NIRS has become a unique and valid tool to facilitate our measurement of LLLT-induced up-regulation of CCO and its relationship to the alteration of hemoglobin oxygenation in treated tissues. We investigated human forearms as a model to reduce tissue heterogeneity during photobiomodulation and to avoid the complication of extra-cerebral layers (i.e., the human scalp and skull). The current study applied LLLT on human forearms using a 1064-nm laser and interleaved the bb-NIRS data acquisition in vivo for simultaneous assessment of interplay between photoactivation/up-regulation of CCO and alteration of hemoglobin oxygenation of the treated tissue.

Results

A total of 11 normal subjects (seven males and four females, mean?±?SD age?=?26.1?±?5.0 years) participated in the experiments. Figure 1 shows dose-dependent (energy density dose?=?exposure time?×?laser power density) concentration changes in [HbO], [Hb], and [CCO] induced by the LLLT and placebo treatments at the group level (mean?±?SE, n?=?11). Overall, LLLT significantly increased the HbO and CCO concentrations as compared with placebo (0.01?<?p?<?0.05 and p?<?0.01), while the Hb concentration was nearly unaltered by either placebo or laser treatment. However, the initial laser effect on CCO seemed to precede in time the effect on HbO; i.e., for [HbO] the laser-induced effect was significantly greater than placebo after two minutes of laser treatment [Fig. 1(a)], whereas for [CCO] the significant effect started after one minute [Fig. 1(c)]. Also note that the increased CCO concentration showed a slightly faster recovery trend towards the baseline than HbO after LLLT.

Figure 1

LLLT (red)/placebo (blue)-induced concentration changes of (a) [HbO], (b) [Hb], and (c) [CCO] in human forearms in vivo (mean?±?SE, n?=?11). In each subplot, the pink-shaded region indicates the period of LLLT/placebo

After close inspection of the data in Fig. 1, we reorganized the respective concentrations and replotted them to show the relationship between concentration increases of CCO vs. HbO or Hb, induced by either LLLT or placebo treatment. As shown in Fig. 2, the solid red dots display the relationship of ?[CCO] vs ?[HbO] during LLLT, with a linear fit by the dashed line (with a correlation coefficient of r?=?0.92 and a p value of 0.001), confirming an excellent linear relationship between them. On the other hand, the open red circles, obtained under the placebo treatment, were gathered within a lower ?[CCO] range with no relationship between ?[CCO] and ?[HbO]. The concentration change of Hb also showed no response to the change of CCO, as plotted by the blue squares in Fig 2.

Figure 2

The relationship of concentration changes between CCO vs. HbO or Hb during LLLT and placebo experiment (mean?±?SE, N?=?11).

Discussion

In this placebo-controlled study, we used broadband NIRS to measure the LLLT-induced changes or increases in oxygenated hemoglobin and CCO concentrations in human forearms in vivo. For the first time, we demonstrated that 1064?nm laser can induce significant increases of CCO and HbO concentrations in a dose-dependent manner over time, as compared with placebo treatment. In addition, ?[CCO] and ?[HbO] displayed a clear linear relationship as the dose of LLLT increased. Especially, we carefully measured and quantified the wavelength-dependent DPF factor, DPF(?) as given by Eq. (5), to minimize crosstalk artifacts35,36. To the best of our knowledge, this is the first study to assess the CCO enzyme up-regulation effects of photobiomodulation in human tissues in vivo. These results demonstrate the great potential of bb-NIRS as a non-invasive technology for mechanistic studies and treatment evaluations of LLLT.

Interplay between up-regulation of CCO and hemoglobin oxygenation induced by LLLT

The observed linearity between ?[CCO] and ?[HbO] induced by LLLT is of great significance, showing a close interplay between the up-regulation of CCO and corresponding hemodynamic oxygenation in the treated tissue. As compared to placebo, the infrared laser treatment induced a significant increase in [CCO] that preceded the increase in [HbO]. Together these data suggest that laser-induced CCO up-regulation leads to a linear increase in HbO. This may indicate that a hemodynamic oxygenation response occurs in vivo as a result of up-regulation of CCO induced by the infrared laser treatment. The mechanism of the observed effects can be explained based on what is known about the role of CCO on photobiomodulation2,39 and the three main steps in cell respiration: glycolysis, Krebs cycle and the electron transport chain. During the first two steps, comparatively little amount of ATP is synthesized. High energy electrons are stored in NADH/FADH; CO2 and water are produced as waste products. In the electron transport chain, where the most amount of energy is produced, CCO (as the terminal enzyme) transfers electrons to enable an oxygen molecule to combine with protons and form a water molecule. At the same time, this process accompanies ATP synthesis (oxidative phosphorylation). Since CCO is the main photo-acceptor within the effective optical window of LLLT, up-regulation of CCO will boost the electron transport, up-regulate the enzymatic activity, and result in a significant increase in oxygen consumption rate within tissue mitochondria. Consequently, an increase in hemodynamic oxygen supply and total blood volume will occur around the LLLT area due to the need for more oxygen and electrons. Therefore, during LLLT, the more the redox state of CCO is activated, the more the oxygenated hemoglobin concentration, HbO, increases proportionally.

In a previous study on the hemodynamic effects of transcranial LLLT on the human brain using dual-wavelength NIRS28, we have reported that transcranial 1064?nm laser improved cerebral oxygenation as indicated by an increase of [HbO] and a decrease of [Hb]. In the current study, we have consistently observed LLLT-induced increases of [HbO] in the human forearms, but [Hb] remained nearly unchanged. A couple of factors may be attributed to this discrepancy in the alteration of [Hb] between the two studies. The first factor can result from the large difference in anatomy and physiology between the human forearm and the brain. The brain is much more vascularized than the forearm, and it has a much greater rate of tissue oxygenation. This may lead to a greater differential hemoglobin concentration in the LLLT-stimulated brain region, with a relative [HbO] increase and [Hb] decrease [24]. The second factor may stem from differences in experimental setups and quantification algorithms that were used to measure and quantify changes in [HbO] and [Hb]. In our previous study, a dual-wavelength NIRS system was used. In consequence, changes in [HbO] and [Hb] were determined using the dual-wavelength-based, modified Beer-Lambert law with a fixed pathlength factor (i.e., DPF is independent of wavelength)28. While the use of a constant DPF is a common practice in the NIRS field, the derived quantifications of ?[HbO] and ?[Hb] are more likely subject to cross-talk errors due to the inaccurate assumption of constant pathlength32,33, particularly when changes of [CCO] are involved. In the current study, on the other hand, we employed a bb-NIRS system, carefully determined wavelength-dependent DPF values, and fitted the measured optical density spectra with a more rigorous expression of Eq. (4) using linear regression analysis. All of these procedures, in principle, should minimize cross-talk errors and lead to improved accuracy of ?[HbO] and ?[Hb] determination35,37. A limitation of this study for transcranial LLLT applications is that the LLLT-induced linear interplay between ?[CCO] and ?[HbO] was demonstrated on the human forearm, but not on the brain. Our future work plans to follow similar LLLT experimental protocols and perform bb-NIRS measurements on the human forehead in order to confirm in the brain the findings reported in this paper.

Rationale of using 1064-nm laser for photobiomodulation

It is noteworthy that the CCO enzyme effects of LLLT are dependent on the wavelength of the stimulation laser (light). A previous study on cultured neurons2 has shown that the most effective wavelengths paralleled the near-infrared absorption peaks of CCO. The current study used a 1064-nm laser that was also employed in our previous studies11,12,28. This wavelength may not be optimal for photon absorption by CCO because its known peaks of light absorption are at lower wavelengths39. However, none of the previous absorption studies have measured photon absorption by CCO at 1064?nm, and the present study demonstrated a clear effect of this wavelength on CCO up-regulation. The primary reason for selecting this wavelength is its ability to better penetrate the human scalp and skull in transcranial applications. In biological tissues, light scattering is the dominant light-tissue interaction, and its influence is two orders of magnitude greater than that of light absorption. According to Mie theory, the light scattering in biological tissues decreases with longer wavelengths40. The 1064-nm wavelength used in our studies is approximately the longest one in the near-infrared optical window where water absorption remains low40. Therefore, 1064?nm is expected to have better penetration depth and stimulation efficiency in the human brain than shorter wavelengths in transcranial laser treatments. For more details on justification of using a 1064-nm laser, please refer to Supplementary Information Section 1.

Measurement accuracy of bb-NIRS on CCO quantification

Quantification of the redox state of cytochrome oxidase in living tissue has been a scientific topic and continuously studied over the last 20+ years30,31,33,35,37,38,41. In particular, continuous development on measurement techniques and improved algorithms have been done by the research group from the University College London, which has continuously made significant efforts to validate and improve the sensitivity, specificity, and accuracy of quantified CCO sensed in living tissues. The listed references of?33,35,37,38,41demonstrate and support the scientific basis and rigor for CCO quantification by bb-NIRS that we utilized in this manuscript.

A possible question is whether, besides CCO, there is any other target or biomarker at the cellular level that contributes to photobiomodulation by absorbing 1064?nm laser. With the current scientific knowledge available, our answer is “No”, which is based on many scientific observations reported in the last 20 or more years. A recent review paper summarized that the major biological tissues that absorb light in 700–1200?nm are blood, water, melanin, adipose tissue/fat, and yellow pigments40. On the other hand, we have not found much reporting in the literature that other LLLT biomarkers at the cellular level (besides CCO) absorb light at 1064?nm in vivo. Future scientific discovery may alter our current view, but the conclusion given in this paper holds its scientific foundation and rigor.

Possible thermal effects of LLLT on CCO quantifications

It is reasonable to expect that infrared light at 1064?nm with a power of 3.4?W would generate some thermal effect that may lead to an increase in skin blood flow (SBF). Such an increase of SBF may give rise to an increase of hemoglobin concentration in the adjacent area surrounding or near the LLLT stimulation spot. To address this concern, we conducted a pilot study, including 4 out of the 11 subjects who participated in the LLLT/Placebo study. Please refer to Supplementary Information Section 2 and Figures for details regarding the results of this pilot study. The clear observation was that thermally induced ?[HbO] followed a similar trend to that of the placebo trace, while the LLLT-induced ?[HbO] remained significantly higher during and after the 8 continuous laser treatments. On the other hand, in the case of ?[CCO], the thermal effect was non-significant on changes in CCO, while significant increases of CCO were clearly observed due to LLLT stimulations.

The overall conclusion from the pilot thermal test was that thermal effects on skin surface may be non-significant to cause changes in tissue redox CCO concentrations that are measured by bb-NIRS with a separation larger than 1.5?cm. However, our sample size for the pilot study was only 4, so this conclusion was not statistically solid, and further studies with more participants are highly desirable to confirm this finding.

In final conclusion, this study has clearly demonstrated that LLLT can induce significant increases of [CCO] and [HbO] on the human forearm as the laser energy dose is accumulated over time, as compared with the placebo treatments. A strong linear interplay between ?[CCO] and ?[HbO] was observed for the first time during the laser treatment, indicating a hemodynamic response of oxygen supply coupled to the increase of cellular metabolic rate induced by photobiomodulation. These results demonstrate the tremendous potential of bb-NIRS as a non-invasive optical means to study in vivo mechanisms and perform treatment evaluations of LLLT.

Material and Methods

Participants

Healthy human participants were recruited from the local community of The University of Texas at Arlington. Interested individuals were screened by one of the investigators to determine whether they were eligible for the study. The inclusion criteria included: either sex, any ethnic background, and in an age range of 18–40 years old. The exclusion criteria included: (1) diagnosed with a psychiatric disorder, (2) history of a neurological condition, or severe brain injury, or violent behavior, (3) have ever been institutionalized/imprisoned, (4) current intake of any medicine or drug, or (5) currently pregnant. In addition, none of the participants were smokers or had diabetes. Eligible participants underwent two separate experiments in sequence: in the first experiment, placebo treatment was administered on their right forearms. In the second experiment, LLLT was administered on the same location as in the placebo treatment, 5?min after the first experiment. The study protocol was approved by the institutional review board (IRB) at The University of Texas at Arlington and complied with all applicable federal and NIH guidelines. Informed consent was obtained from each participant prior to the experiments.

Instruments

Both placebo and laser treatments were administered with a continuous-wave, 1064-nm laser provided by Cell Gen Therapeutics LLC, Dallas, TX (Model CG-5000). This laser is an FDA-cleared device for various uses on humans, such as relief of muscle and joint pain. It had a hand-held aperture with a button on the handle to open and shut the laser beam. The area of laser beam from the aperture was 13.6?cm2. Contact delivery is relevant when laser beams are divergent and not well collimated. But in our case, the laser was well collimated, so the laser beam size did not change significantly between the laser aperture and the stimulation spot on the subject’s forearm. The non-contact delivery distance was about 2?cm with possible variation of a few millimeters because of the handheld setting. However, such a distance variation did not result in dose fluctuation in laser radiation due to excellent laser collimation. For the laser treatment, the device was operated at a constant power of 3.4?W. The irradiance (or power density) in the beam area was 0.25?W/cm2, the same as that used in our previous studies11,12,28. For the placebo treatment, the same device was operated at a minimal power of 0.1?W and the aperture was further covered up by black tapes so that no light came out from the covering tapes. Thus the actual laser power of placebo was zero.

While we used a FDA-cleared Class 4 infrared laser (International standard IEC 60825-1), this laser was used at a lower power density corresponding to that of a Class 3b laser to avoid potential skin damage. The power density used was 0.25?W/cm2 (whereas over 0.5?W/cm2 is used for Class 4 classification). Following previously successful studies with full IRB approvals, our safe laser stimulation parameters were calculated as follows:

Total laser power?=?3.4?W;

Area of laser beam radiation?=?13.6?cm2;

Power density?=?3.4?W/13.6?cm2?=?0.25?W/cm2;

Time radiated per cycle?=?55?s;

Total laser energy dose per cycle?=?3.4?W?×?55?s?=?187?J/cycle.

If another laser with a smaller beam size is used, the total laser power should be adjusted in order to maintain the same safe low power density of 0.25?W/cm2 and thus avoid potential skin damage.

A single-channel, bb-NIRS system was constructed to measure changes of hemoglobin and CCO concentrations in vivo in LLLT and placebo experiments. As shown in Fig. 3, this system consisted of a tungsten halogen lamp (Model 3900, Illumination Technologies Inc., East Syracuse, NY) as light source and a miniature back-thinned CCD spectrometer (i-trometer, B&W Tek Inc., Newark, DE) as light detector, in the spectral range of 450–1100?nm. Broadband white light from the lamp was relayed by an optical fiber bundle of 3.5-mm in diameter to a shutter and then to an I-shaped optical probe holder that was placed on each subject’s right forearm. The diffuse light through the arm tissue was collected by another fiber bundle held by the same probe holder and then relayed to the spectrometer. The distance between the source and detector fiber bundles was 1.5?cm. A laptop computer was used to acquire, display and save the data from the spectrometer. The shutter controlled the on and off of the white light delivered to the tissues.

Figure 3

Schematic diagram of the experimental setup, including the broadband NIRS system.

In particular, the I-shaped fiber bundle holder was designed using SolidWorks (SolidWorks Corp., USA) and 3D printed with solid, black material. The two wider ends of the holder were firmly fastened on each participant’s right forearm with elastic bandages (see Fig. 4a). As each participant might have slight body movements during the corresponding experiment, this experimental setup minimized potential motion artifacts during data acquisition. The narrow, middle section of the holder is ~8?mm in width. In both experiments, the laser beam from CG-5000 was administered on both sides of this section alternatively (see Fig. 4b).

Figure 4

Experimental setup: (a) photograph of the laser aperture for LLLT/placebo treatment and bb-NIRS fiber holder on a participant’s forearm. (b) Configuration of the I-shaped bb-NIRS probe holder (dark gray). The bundle holder held two optical fiber

Experiments

The experiments were conducted in a locked room without any reflective surface. The background light from outside of the room was minimized by covering the windows and door slits with black curtains. Furthermore, when the laser was in use, a warning sign of “Laser on” was shown on the outer door. Protective goggles (900–1000?nm: 5+, 1000–2400?nm: 7+; 2900–10600?nm: 7+) were worn by all individuals present in the room. The participants were further instructed to close eyes during the treatments. After each participant was comfortably seated, an experimenter first measured the absorption coefficient (?a) and reduced scattering coefficient (?s?) at 750?nm and 830?nm from the participant’s right forearm using a frequency-domain NIRS tissue oximeter (OxiplexTS, ISS Inc., Champaign, IL). Then the I-shaped optical probe holder was placed on the same location. A trained experimenter held the aperture of CG-5000 laser closely to the participant’s right forearm to administer the placebo or LLLT treatment on two sides of the holder alternatively. As illustrated inFig. 5, each treatment session consisted of eight one-minute cycles, 55-s laser on and 5-s laser off per cycle. The participant was given a 2-minute break between the two experiments.

Figure 5

Paradigm of the LLLT/placebo treatment and interleaved bb-NIRS data acquisition.

The participants received no information about the treatment type (placebo or LLLT) in each experiment. Instead, they were instructed that they would receive the same laser treatment at a power of 3.4?W in both experiments. Furthermore, the laser at a power of 3.4?W generated negligible heat on the participants’ skin. Thus, the two experiments were designed to cause approximately the same sensations and expectations in the participants.

The data acquisition of bb-NIRS was initiated two minutes before each treatment session and ceased five minutes after the treatment session. Because the power of treatment laser from CG-5000 was high enough to contaminate the bb-NIRS readings, data acquisition interleaved with the treatment cycles during the 5-s laser-off periods. Following the similar format/fashion, the data during pre-treatment baseline and post-treatment recovery were also acquired at 55-second interval. In this way, a total of 15 data points (see Fig. 5) were obtained throughout each experiment. The shutter was switched on only for 5 seconds during each data acquisition period and then off in the rest of the time.

Theoretical Foundation for Data Processing and Error Analysis

Raw data from the broadband spectrometer was processed using MATLAB to calculate relative changes in [HbO], [Hb] and [CCO] from the initial baseline. First, the relative optical density, ?OD, was calculated at each wavelength, ?:

An external file that holds a picture, illustration, etc.
Object name is srep30540-m1.jpg

where I0(?) is the spectral data acquired at the initial baseline (i.e., the first spectrum collected in each experiment), and I(?) is the data acquired at each time point thereafter.

According to the Modified Beer-Lambert Law21,42, ?OD at each ? could be expressed as a sum of optical absorbance contributed by HbO, Hb and CCO components:

An external file that holds a picture, illustration, etc.
Object name is srep30540-m2.jpg

where ?[HbO] is the relative change in HbO concentration, ?[Hb] is the relative change in Hb concentration, ?[CCO] is the relative change in CCO concentration, ?HbO (?), ?Hb(?) and ?CCO(?) are the extinction coefficients of HbO, Hb and CCO, which can be found in ref. 35, and L(?) denotes the effective pathlength of the detected photons through the tissues. According to the Modified Beer-Lambert Law21,42,L(?) can be estimated as:

An external file that holds a picture, illustration, etc.
Object name is srep30540-m3.jpg

where r is the source-detector distance, and DPF(?) is the wavelength-dependent differential pathlength factor. Note that in this study, we did not assume that DPF was a wavelength-independent constant across the wavelength range. By substituting Eq. (3) into Eq. (2) for multiple wavelengths, we can express ?[HbO], ?[Hb] and ?[CCO] in a matrix format in association with broadband ?OD(?) over DPF(?), as follows:

An external file that holds a picture, illustration, etc.
Object name is srep30540-m4.jpg

In this study, the DPF(?) values were estimated based on the ?a and ?s? values measured with a frequency-domain OxiplexTS tissue oximeter in the beginning of the experiments. In principle, an OxiplexTS tissue oximeter provides measurement readings of ?a and ?s? values at 750?nm and 830?nm as well as absolute concentrations of [HbO] and [Hb]. To achieve ?a and ?s? values across the entire wavelength range of 740–900?nm, we interpolated and extrapolated the two measured ?s? values at 750?nm and 830?nm by following Mie theory, which is usually expressed by k??b, where k and b were obtained by fitting this equation to both?s?(750?nm) and ?s?(830?nm). In the meantime, the absorption coefficients in the same wavelength range (740–900?nm) were estimated based on the HbO and Hb concentrations measured by the same tissue oximeter. Then, the wavelength-dependent DPF values were calculated using the diffusion theory with the semi-infinite boundary geometry43:

An external file that holds a picture, illustration, etc.
Object name is srep30540-m5.jpg

where ?a(?) and ?s?(?) are the estimated absorption and reduced scattering coefficients across the wavelength range of interest.

Next, the final and key step was to quantify or determine three chromophore concentrations based on Eq. (4). To do so, multiple linear regression analysis was implemented in the wavelength range of 740–900?nm (with a total of 161 wavelengths) using a MATLAB-based function. This regression algorithm afforded the best fit of the chromophore-specific concentrations to the measured ?OD(?) spectrum by minimizing the squared residual or the objective function. The detailed fitting procedures are given next.

Multiple Linear Regression Analysis

The procedure for multiple linear regression analysis is outlined with a flow chart in Fig. 6.(1)

Figure 6

A flow chart describing detailed procedures of our multiple linear regression analysis to optimally determine LLLT-induced concentration changes in three chromophores.

(1) Start data collection and quantification of ?a and ?s? values at 750?nm and 830?nm of the subject’s right forearm by the OxiplexTS tissue oximeter.(2)

(2) Obtain output readings from the oximeter for absolute concentrations of [HbO] and [Hb].(3)

(3) Interpolate and extrapolate ?a(?) and ?s?(?) values across 740?nm to 900?nm, based on ?a and ?s? values at 750?nm and 830?nm, by fitting Mie theory (k??b) and calculating [HbO] and [Hb] with their corresponding extinction coefficients given in ref. 35.(4)

(4) Calculate DPF(?) values across 740?nm to 900?nm for each wavelength with Eq. (5);(5)

(5) Perform bb-NIRS experiments to acquire optical spectra (with 161 wavelengths from 740–900?nm) from the subject’s arm before, during and after LLLT (or placebo);(6)

(6) Quantifying ?OD(?) at different time points, using Eq. (1), to form a time series for each wavelength.(7)

(7) Build up or form the 161×3 extinction coefficient matrix, based on ref. 35; this matrix would list extinction coefficients at 161 wavelengths (740–900?nm) for 3 chromophores.(8)

(8) Solve accurately ?[HbO], ?[Hb] and ?[CCO] based on Eq. (4). For the last step, specifically, we applied the MATLAB function of “fminsearch” to find/fit for the optimal combination of ?[HbO], ?[Hb] and ?[CCO] as the final output parameters. In theory, “fminsearch” function finds the minimum of a scalar function (often called the objective function) of several variables, starting at an initial estimate (http://www.mathworks.com/help/optim/ug/fminsearch.html). This is generally referred to as unconstrained nonlinear optimization. In our study, for example, “fminsearch” attempted to model the relationship between three explanatory variables (e.g., ?[HbO], ?[Hb] and ?[CCO]) and a set of response variables (e.g., ?OD at multi-wavelengths) by fitting a linear equation to observed data, as expressed in Eq. (4). This function tries different combination of ?[HbO], ?[Hb] and ?[CCO] starting from the “initial guess” to match with the measured set of ?OD(?) across the spectra until a minimized value of the objective equation is achieved. Then, this set of values (?[HbO], ?[Hb] and ?[CCO]) are considered as the “best fit” and to be used as the final results.

Statistical Analysis

To determine whether the LLLT induced significant changes in hemoglobin and CCO concentrations with respect to the placebo treatment, paired t-test between these two treatment types was conducted for each chromophore (HbO, Hb and CCO) at each time point. A two-tailed level of 0.01?<?p?<?0.05 and p?<?0.01 was chosen to be statistically significant in these tests.

Additional Information

How to cite this article: Wang, X. et al. Interplay between up-regulation of cytochrome-c-oxidase and hemoglobin oxygenation induced by near-infrared laser. Sci. Rep. 6, 30540; doi: 10.1038/srep30540 (2016).

Supplementary Material

Supplementary Information:

Acknowledgments

Dr. Gonzalez-Lima reported receiving support from an institutional research fellowship from the College of Liberal Arts of the University of Texas at Austin. Dr. Gonzalez-Lima holds the George I. Sanchez Centennial Endowed Professorship in Liberal Arts and Sciences. The authors acknowledge the support in part from the University of Texas BRAIN Initiative Seed Funding (#362718).

Footnotes

Author Contributions X.W. performed the experiment, developed data analysis algorithms, analyzed the data and prepared/wrote the manuscript. F.T. designed the experiment, discussed and interpreted the results, and participated in manuscript writing-up and revision. S.S.S. participated in performing the experiment. F.G.-L. initiated the study with H.L., discussed and interpreted the results, and participated in manuscript revision. H.L. initiated and supervised the study, discussed and interpreted the results, as well as reviewed and revised the manuscript.

References

  • Eells J. T. et al. . Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy. Mitochondrion 4, 559–567, (2004).10.1016/j.mito.2004.07.033 [PubMed][Cross Ref]
  • Wong-Riley M. T. et al. . Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase. J Biol Chem 280, 4761–4771, (2005).10.1074/jbc.M409650200 [PubMed] [Cross Ref]
  • Chow R. T., Johnson M. I., Lopes-Martins R. A. & Bjordal J. M. Efficacy of low-level laser therapy in the management of neck pain: a systematic review and meta-analysis of randomised placebo or active-treatment controlled trials. Lancet 374, 1897–1908, (2009).10.1016/S0140-6736(09)61522-1[PubMed] [Cross Ref]
  • Kingsley J. D., Demchak T. & Mathis R. Low-level laser therapy as a treatment for chronic pain. Front Physiol 5, 306, (2014).10.3389/fphys.2014.00306 [PMC free article] [PubMed] [Cross Ref]
  • Lampl Y. et al. . Infrared laser therapy for ischemic stroke: a new treatment strategy: results of the NeuroThera Effectiveness and Safety Trial-1 (NEST-1). Stroke; a journal of cerebral circulation 38, 1843–1849, (2007).10.1161/STROKEAHA.106.478230 [PubMed] [Cross Ref]
  • Zivin J. A. et al. . Effectiveness and safety of transcranial laser therapy for acute ischemic stroke.Stroke 40, 1359–1364, (2009).10.1161/STROKEAHA.109.547547 [PubMed] [Cross Ref]
  • Naeser M. A., Saltmarche A., Krengel M. H., Hamblin M. R. & Knight J. A. Improved cognitive function after transcranial, light-emitting diode treatments in chronic, traumatic brain injury: two case reports. Photomed Laser Surg 29, 351–358, (2011).10.1089/pho.2010.2814 [PMC free article][PubMed] [Cross Ref]
  • Naeser M. A. et al. . Significant improvements in cognitive performance post-transcranial, red/near-infrared light-emitting diode treatments in chronic, mild traumatic brain injury: open-protocol study. J Neurotrauma 31, 1008–1017, (2014).10.1089/neu.2013.3244 [PMC free article] [PubMed][Cross Ref]
  • Schiffer F. et al. . Psychological benefits 2 and 4 weeks after a single treatment with near infrared light to the forehead: a pilot study of 10 patients with major depression and anxiety. Behavioral and brain functions : BBF 5, 46, (2009).10.1186/1744-9081-5-46 [PMC free article] [PubMed] [Cross Ref]
  • Disner S. G., Beevers C. G. & Gonzalez-Lima F. Transcranial Laser Stimulation as Neuroenhancement for Attention Bias Modification in Adults with Elevated Depression Symptoms.Brain Stimul , (2016).10.1016/j.brs.2016.05.009 [PubMed] [Cross Ref]
  • Barrett D. W. & Gonzalez-Lima F. Transcranial infrared laser stimulation produces beneficial cognitive and emotional effects in humans. Neuroscience 230, 13–23, (2013).10.1016/j.neuroscience.2012.11.016 [PubMed] [Cross Ref]
  • Blanco N. J., Maddox W. T. & Gonzalez-Lima F. Improving executive function using transcranial infrared laser stimulation. Journal of neuropsychology , (2015).10.1111/jnp.12074 [PMC free article][PubMed] [Cross Ref]
  • Hwang J., Castelli D. M. & Gonzalez-Lima F. Cognitive enhancement by transcranial laser stimulation and acute aerobic exercise. Lasers Med Sci , (2016).10.1007/s10103-016-1962-3 [PubMed][Cross Ref]
  • Pastore D., Greco M. & Passarella S. Specific helium-neon laser sensitivity of the purified cytochrome c oxidase. Int J Radiat Biol 76, 863–870 (2000). [PubMed]
  • Gonzalez-Lima F. & Auchter A. Protection against neurodegeneration with low-dose methylene blue and near-infrared light. Front Cell Neurosci 9, 179, (2015).10.3389/fncel.2015.00179[PMC free article] [PubMed] [Cross Ref]
  • Gonzalez-Lima F., Barksdale B. R. & Rojas J. C. Mitochondrial respiration as a target for neuroprotection and cognitive enhancement. Biochemical pharmacology 88, 584–593, (2014).10.1016/j.bcp.2013.11.010 [PubMed] [Cross Ref]
  • Rojas J. C. & Gonzalez-Lima F. Neurological and psychological applications of transcranial lasers and LEDs. Biochemical pharmacology 86, 447–457, (2013).10.1016/j.bcp.2013.06.012 [PubMed][Cross Ref]
  • Gonzalez-Lima F. & Barrett D. W. Augmentation of cognitive brain functions with transcranial lasers.Frontiers in systems neuroscience 8, 36, (2014).10.3389/fnsys.2014.00036 [PMC free article][PubMed] [Cross Ref]
  • Rojas J. C., Bruchey A. K. & Gonzalez-Lima F. Low-level light therapy improves cortical metabolic capacity and memory retention. Journal of Alzheimer’s disease: JAD 32, 741–752, (2012).10.3233/JAD-2012-120817 [PubMed] [Cross Ref]
  • Quaresima V., Bisconti S. & Ferrari M. A brief review on the use of functional near-infrared spectroscopy (fNIRS) for language imaging studies in human newborns and adults. Brain Lang 121, 79–89, (2012).10.1016/j.bandl.2011.03.009 [PubMed] [Cross Ref]
  • Scholkmann F. et al. . A review on continuous wave functional near-infrared spectroscopy and imaging instrumentation and methodology. NeuroImage 85 Pt 1, 6–27, (2014).10.1016/j.neuroimage.2013.05.004 [PubMed] [Cross Ref]
  • Cerussi A. E. et al. . Diffuse optical spectroscopic imaging correlates with final pathological response in breast cancer neoadjuvant chemotherapy. Philos Trans A Math Phys Eng Sci 369, 4512–4530, (2011).10.1098/rsta.2011.0279 [PMC free article] [PubMed] [Cross Ref]
  • Jiang S. et al. . Pilot study assessment of dynamic vascular changes in breast cancer with near-infrared tomography from prospectively targeted manipulations of inspired end-tidal partial pressure of oxygen and carbon dioxide. Journal of biomedical optics 18, 76011, (2013).10.1117/1.JBO.18.7.076011[PMC free article] [PubMed] [Cross Ref]
  • Durduran T., Choe R., Baker W. B. & Yodh A. G. Diffuse Optics for Tissue Monitoring and Tomography. Rep Prog Phys 73, (2010).10.1088/0034-4885/73/7/076701 [PMC free article][PubMed] [Cross Ref]
  • Jiang Z. et al. . Trans-rectal ultrasound-coupled near-infrared optical tomography of the prostate, part II: experimental demonstration. Optics express 16, 17505–17520 (2008). [PubMed]
  • Jiang Z. et al. . Trans-rectal ultrasound-coupled spectral optical tomography of total hemoglobin concentration enhances assessment of the laterality and progression of a transmissible venereal tumor in canine prostate. Urology 77, 237–242, (2011).10.1016/j.urology.2010.06.017 [PubMed][Cross Ref]
  • Boas D. A., Elwell C. E., Ferrari M. & Taga G. Twenty years of functional near-infrared spectroscopy: introduction for the special issue. NeuroImage 85 Pt 1, 1–5, (2014).10.1016/j.neuroimage.2013.11.033[PubMed] [Cross Ref]
  • Tian F., Hase S. N., Gonzalez-Lima F. & Liu H. Transcranial laser stimulation improves human cerebral oxygenation. Lasers Surg Med , (2016).10.1002/lsm.22471 [PubMed] [Cross Ref]
  • Ferrari M. a. Q., V. Near infrared brain and muscle oximetry: from the discovery to current applications. Journal of Near Infrared Spectroscopy 20, 1–14 (2012).
  • Matcher S. J., Elwell C. E., Cooper C. E., Cope M. & Delpy D. T. Performance comparison of several published tissue near-infrared spectroscopy algorithms. Anal Biochem 227, 54–68, (1995).10.1006/abio.1995.1252 [PubMed] [Cross Ref]
  • Gagnon R. E., Gagnon F. A. & Macnab A. J. Comparison of 13 published cytochrome c oxidase near-infrared spectroscopy algorithms. Eur J Appl Physiol Occup Physiol 74, 487–495 (1996). [PubMed]
  • Uludag K., Kohl M., Steinbrink J., Obrig H. & Villringer A. Cross talk in the Lambert-Beer calculation for near-infrared wavelengths estimated by Monte Carlo simulations. Journal of biomedical optics 7, 51–59, (2002).10.1117/1.1427048 [PubMed] [Cross Ref]
  • Uludag K. et al. . Cytochrome-c-oxidase redox changes during visual stimulation measured by near-infrared spectroscopy cannot be explained by a mere cross talk artefact. NeuroImage 22, 109–119, (2004).10.1016/j.neuroimage.2003.09.053 [PubMed] [Cross Ref]
  • Tachtsidis I., Koh P. H., Stubbs C. & Elwell C. E. Functional optical topography analysis using statistical parametric mapping (SPM) methodology with and without physiological confounds. Adv Exp Med Biol 662, 237–243, (2010).10.1007/978-1-4419-1241-1_34 [PMC free article] [PubMed][Cross Ref]
  • Kolyva C. et al. . Systematic investigation of changes in oxidized cerebral cytochrome c oxidase concentration during frontal lobe activation in healthy adults. Biomedical optics express 3, 2550–2566, (2012).10.1364/BOE.3.002550 [PMC free article] [PubMed] [Cross Ref]
  • Kolyva C. et al. . Cytochrome c oxidase response to changes in cerebral oxygen delivery in the adult brain shows higher brain-specificity than haemoglobin. NeuroImage 85 Pt 1, 234–244, (2014).10.1016/j.neuroimage.2013.05.070 [PMC free article] [PubMed] [Cross Ref]
  • Bale G., Mitra S., Meek J., Robertson N. & Tachtsidis I. A new broadband near-infrared spectroscopy system for in-vivo measurements of cerebral cytochrome-c-oxidase changes in neonatal brain injury.Biomedical optics express 5, 3450–3466, (2014).10.1364/BOE.5.003450 [PMC free article][PubMed] [Cross Ref]
  • Bainbridge A. et al. . Brain mitochondrial oxidative metabolism during and after cerebral hypoxia-ischemia studied by simultaneous phosphorus magnetic-resonance and broadband near-infrared spectroscopy. NeuroImage 102 Pt 1, 173–183, (2014).10.1016/j.neuroimage.2013.08.016[PMC free article] [PubMed] [Cross Ref]
  • Karu T. I., Pyatibrat L. V., Kolyakov S. F. & Afanasyeva N. I. Absorption measurements of a cell monolayer relevant to phototherapy: reduction of cytochrome c oxidase under near IR radiation. J Photochem Photobiol B 81, 98–106, (2005).10.1016/j.jphotobiol.2005.07.002 [PubMed] [Cross Ref]
  • Jacques S. L. Optical properties of biological tissues: a review. Phys Med Biol 58, R37–61, (2013).10.1088/0031-9155/58/11/R37 [PubMed] [Cross Ref]
  • Tachtsidis I. et al. . A hybrid multi-distance phase and broadband spatially resolved spectrometer and algorithm for resolving absolute concentrations of chromophores in the near-infrared light spectrum.Adv Exp Med Biol 662, 169–175, (2010).10.1007/978-1-4419-1241-1_24 [PMC free article][PubMed] [Cross Ref]
  • Kocsis L., Herman P. & Eke A. The modified Beer-Lambert law revisited. Phys Med Biol 51, N91–98, (2006).10.1088/0031-9155/51/5/N02 [PubMed] [Cross Ref]
  • Fantini S. et al. . Non-invasive optical monitoring of the newborn piglet brain using continuous-wave and frequency-domain spectroscopy. Phys Med Biol 44, 1543–1563 (1999). [PubMed]

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group
Sci Rep. 2016 Aug 3;6:30540. doi: 10.1038/srep30540.

Interplay between up-regulation of cytochrome-c-oxidase and hemoglobin oxygenation induced by near-infrared laser.

Wang X1, Tian F1, Soni SS1, Gonzalez-Lima F2, Liu H1.

Author information

  • 1Department of Bioengineering, the University of Texas at Arlington, 500 UTA Blvd, Arlington, TX 76010, USA.
  • 2Department of Psychology and Institute for Neuroscience, the University of Texas at Austin, 108 E. Dean Keeton Stop A8000, Austin, TX 78712, USA.

Abstract

Photobiomodulation, also known as low-level laser/light therapy (LLLT), refers to the use of red-to-near-infrared light to stimulate cellular functions for physiological or clinical benefits. The mechanism of LLLT is assumed to rely on photon absorption by cytochrome c oxidase (CCO), the terminal enzyme in the mitochondrial respiratory chain that catalyzes the reduction of oxygen for energy metabolism. In this study, we used broadband near-infrared spectroscopy (NIRS) to measure the LLLT-induced changes in CCO and hemoglobin concentrations in human forearms in vivo. Eleven healthy participants were administered with 1064-nm laser and placebo treatments on their right forearms. The spectroscopic data were analyzed and fitted with wavelength-dependent, modified Beer-Lambert Law. We found that LLLT induced significant increases of CCO concentration (Delta[CCO]) and oxygenated hemoglobin concentration (Delta[HbO]) on the treated site as the laser energy dose accumulated over time. A strong linear interplay between Delta[CCO] and [HbO] was observed for the first time during LLLT, indicating a hemodynamic response of oxygen supply and blood volume closely coupled to the up-regulation of CCO induced by photobiomodulation. These results demonstrate the tremendous potential of broadband NIRS as a non-invasive, in vivo means to study mechanisms of photobiomodulation and perform treatment evaluations of LLLT.

Lasers Med Sci. 2014 Jun 24. [Epub ahead of print]

Effect of pre-irradiation with different doses, wavelengths, and application intervals of low-level laser therapy on cytochrome c oxidase activity in intact skeletal muscle of rats.

Albuquerque-Pontes GM1, Vieira RD, Tomazoni SS, Caires CO, Nemeth V, Vanin AA, Santos LA, Pinto HD, Marcos RL, Bjordal JM, de Carvalho PD, Leal-Junior EC.

Author information

  • 1Postgraduate Program in Biophotonics Applied to Health Sciences, Universidade Nove de Julho (UNINOVE), Rua Vergueiro, 235, São Paulo, SP, 01504-001, Brazil.

Abstract

Modulation of cytochrome c oxidase activity has been pointed as a possible key mechanism for low-level laser therapy (LLLT) in unhealthy biological tissues. But recent studies by our research group with LLLT in healthy muscles before exercise found delayed skeletal muscle fatigue development and improved biochemical status in muscle tissue. Therefore, the aim of this study was to evaluate effects of different LLLT doses and wavelengths in cytochrome c oxidase activity in intact skeletal muscle. In this animal experiment, we irradiated the tibialis anterior muscle of rats with three different LLLT doses (1, 3, and 10 J) and wavelengths (660, 830, and 905 nm) with 50 mW power output. After irradiation, the analyses of cytochrome c oxidase expression by immunohistochemistry were analyzed at 5, 10, 30 min and at 1, 2, 12, and 24 h. Our results show that LLLT increased (p?<?0.05) cytochrome c oxidase expression mainly with the following wavelengths and doses: 660 nm with 1 J, 830 nm with 3 J, and 905 nm with 1 J at all time points. We conclude that LLLT can increase cytochrome c oxidase activity in intact skeletal muscle and that it contributes to our understanding of how LLLT can enhance performance and protect skeletal muscles against fatigue development and tissue damage. Our findings also lead us to think that the combined use of different wavelengths at the same time can enhance LLLT effects in skeletal muscle performance and other conditions, and it can represent a therapeutic advantage in clinical settings.

 

J Photochem Photobiol B. 2009 May 4;95(2):89-92. Epub 2009 Jan 21.

Evaluation of mitochondrial respiratory chain activity in muscle healing by low-level laser therapy.

Silveira PC, Silva LA, Fraga DB, Freitas TP, Streck EL, Pinho R.

Laboratório de Fisiologia e Bioquímica do Exercício, Programa de Pós-graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil. silveira_paulo2004@yahoo.com.br

Abstract

BACKGROUND: Recent studies demonstrate that low-level laser therapy (LLLT) modulates many biochemical processes, especially the decrease of muscle injures, the increase in mitochondrial respiration and ATP synthesis for accelerating the healing process. OBJECTIVE: In this work, we evaluated mitochondrial respiratory chain complexes I, II, III and IV and succinate dehydrogenase activities after traumatic muscular injury. METHODS: Male Wistar rats were randomly divided into three groups (n=6): sham (uninjured muscle), muscle injury without treatment, muscle injury with LLLT (AsGa) 5J/cm(2). Gastrocnemius injury was induced by a single blunt-impact trauma. LLLT was used 2, 12, 24, 48, 72, 96, and 120 hours after muscle-trauma. RESULTS: Our results showed that the activities of complex II and succinate dehydrogenase after 5days of muscular lesion were significantly increased when compared to the control group. Moreover, our results showed that LLLT significantly increased the activities of complexes I, II, III, IV and succinate dehydrogenase, when compared to the group of injured muscle without treatment. CONCLUSION: These results suggest that the treatment with low-level laser may induce an increase in ATP synthesis, and that this may accelerate the muscle healing process.

Photomed Laser Surg. 2008 Dec;26(6):593-9.

Absorption measurements of cell monolayers relevant to mechanisms of laser phototherapy: reduction or oxidation of cytochrome c oxidase under laser radiation at 632.8 nm.

Karu TI, Pyatibrat LV, Kolyakov SF, Afanasyeva NI.

Institute of Laser and Information Technologies of Russian Academy of Sciences, Troitsk, Moscow Region 142190, Russian Federation. tkaru@isan.troitsk.ru

Abstract

OBJECTIVE: The objective of this work was a further investigation of redox mechanisms of laser phototherapy on the cellular level.

BACKGROUND DATA: Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is believed to work as the photoacceptor to modulate cellular metabolism in laser phototherapy.

MATERIALS AND METHODS: The changes in the absorption spectra of HeLa-cell monolayers before and after irradiation at 632.8 nm using fast multi-channel recording were evaluated by the intensity ratio between the peaks at 770 and 670 nm (intensity ratio criterion).

RESULTS: By the intensity ratio criterion, the irradiation effects (reduction or oxidation of the photoacceptor) depended on the initial redox status of cytochrome c oxidase. The irradiation (three times at 632.8 nm, dose = 6.3 x 103 J/m(2), tau(irrad.) = 10 sec, tau(record.) = 600 msec) of cells initially characterized by relatively oxidized cytochrome c oxidase caused first a reduction of the photoacceptor, and then its oxidation (a bell-shaped curve). The irradiation by the same scheme of the cells with initially relatively reduced cytochrome c oxidase caused first oxidation and then a slight reduction of the enzyme (a curve opposite to the bell-shaped curve).

CONCLUSION: The experimental results of our work demonstrate that irradiation at 632.8 nm causes either a (transient) relative reduction of the photoacceptor, putatively cytochrome c oxidase, or its (transient) relative oxidation, depending on the initial redox status of the photoacceptor. The maximum in the bell-shaped dose-dependence curve or the minimum of the reverse curve is the turning point between the prevailing of oxidation or reduction processes. Our results are evidence that the bell-shaped dose dependences recorded for various cellular responses are characteristic also for redox changes in the photoacceptor, cytochrome c oxidase.

J Photochem Photobiol B. 2007 Mar 1;86(3):279-82. Epub 2006 Nov 20.

Evaluation of mitochondrial respiratory chain activity in wound healing by low-level laser therapy.

 

Silveira PC, Streck EL, Pinho RA.

Laboratório de Fisiologia e Bioquímica do Exercício, Universidade do Extremo Sul Catarinense, 88806-000 Criciúma, SC, Brazil.

Abstract

Laser therapy is used in many biomedical sciences to promote tissue regeneration. Many studies involving low-level laser therapy have shown that the healing process is enhanced by such therapy. In this work, we evaluated mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase activities in wounds after irradiation with low-level laser. The animals were divided into two groups: group 1, the animals had no local nor systemic treatment and were considered as control wounds; group 2, the wounds were treated immediately after they were made and every day after with a low-level laser (AsGa, wavelength of 904 nm) for 10 days. The results showed that low-level laser therapy improved wound healing. Besides, our results showed that low-level laser therapy significantly increased the activities of complexes II and IV but did not affect succinate dehydrogenase activity. These findings are in accordance to other works, where cytochrome c oxidase (complex IV) seems to be activated by low-level laser therapy. Besides, we showed, for the first time, that complex II activity was also activated. More studies are being carried out in order to evaluate other mitochondrial enzymes activities after different doses and irradiation time of low-level laser.

Photomed Laser Surg. 2006 Apr;24(2):121-8.

Clinical and experimental applications of NIR-LED photobiomodulation.

Desmet KD, Paz DA, Corry JJ, Eells JT, Wong-Riley MT, Henry MM, Buchmann EV, Connelly MP, Dovi JV, Liang HL, Henshel DS, Yeager RL, Millsap DS, Lim J, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT.

Department of Clinical Laboratory Sciences, University of Wisconsin-Milwaukee, 53226, USA.

This review presents current research on the use of far-red to near-infrared (NIR) light treatment in various in vitro and in vivo models. Low-intensity light therapy, commonly referred to as “photobiomodulation,” uses light in the far-red to near-infrared region of the spectrum (630-1000 nm) and modulates numerous cellular functions. Positive effects of NIR-light-emitting diode (LED) light treatment include acceleration of wound healing, improved recovery from ischemic injury of the heart, and attenuated degeneration of injured optic nerves by improving mitochondrial energy metabolism and production. Various in vitro and in vivo models of mitochondrial dysfunction were treated with a variety of wavelengths of NIR-LED light. These studies were performed to determine the effect of NIR-LED light treatment on physiologic and pathologic processes. NIRLED light treatment stimulates the photoacceptor cytochrome c oxidase, resulting in increased energy metabolism and production. NIR-LED light treatment accelerates wound healing in ischemic rat and murine diabetic wound healing models, attenuates the retinotoxic effects of methanol-derived formic acid in rat models, and attenuates the developmental toxicity of dioxin in chicken embryos. Furthermore, NIR-LED light treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. The experimental results demonstrate that NIR-LED light treatment stimulates mitochondrial oxidative metabolism in vitro, and accelerates cell and tissue repair in vivo. NIR-LED light represents a novel, noninvasive, therapeutic intervention for the treatment of numerous diseases linked to mitochondrial dysfunction.

J Photochem Photobiol B. 2005 Nov 1;81(2):98-106. Epub 2005 Aug 26.

Absorption measurements of a cell monolayer relevant to phototherapy: reduction of cytochrome c oxidase under near IR radiation.

Karu TI, Pyatibrat LV, Kolyakov SF, Afanasyeva NI.

Institute of Laser and Information Technologies of Russian Academy of Sciences, Troitsk, Pionerskaya Street 2, Moscow Region 142190, Russian Federation. tkaru@isan.troitsk.ru

Phototherapy uses monochromatic light in the optical region of 600-1000 nm to treat in a non-destructive and non-thermal fashion various soft-tissue and neurological conditions. This kind of treatment is based on the ability of light red-to-near IR to alter cellular metabolism as a result of its being absorbed by cytochrome c oxidase. To further investigate the involvement of cytochrome c oxidase as a photoacceptor in the alteration of the cellular metabolism, we have aimed our study at, first, recording the absorption spectra of HeLa-cell monolayers in various oxygenation conditions (using fast multichannel recording), secondly, investigating the changes caused in these absorption spectra by radiation at 830 nm (the radiation wavelength often used in phototherapy), and thirdly, comparing between the absorption and action spectra recorded. The absorption measurements have revealed that the 710- to 790-nm spectral region is characteristic of a relatively reduced photoacceptor, while the 650- to 680-nm one characterizes a relatively oxidized photoacceptor. The ratio between the peak intensities at 760 and 665 nm is used to characterize the redox status of cytochrome c oxidase. By this criterion, the irradiation of the cellular monolayers with light at lambda=830 nm (D=6.3 x 10(3)J/m(2)) causes the reduction of the photoacceptor. A similarity is established between the peak positions at 616, 665, 760, 813, and 830 nm in the absorption spectra of the cellular monolayers and the action spectra of the long-term cellular responses (increase in the DNA synthesis rate and cell adhesion to a matrix).

Photomed Laser Surg. 2005 Aug;23(4):355-61.

Exact action spectra for cellular responses relevant to phototherapy.

Karu TI, Kolyakov SF.

Institute of Laser and Information Technologies, Russian Academy of Sciences, Troitsk, Moscow Region, Russian Federation. tkaru@isan.troitsk.ru

OBJECTIVE: The aim of the present work is to analyze available action spectra for various biological responses of HeLa cells irradiated with monochromatic light of 580-860 nm.

BACKGROUND DATA: Phototherapy (low-level laser therapy or photobiomodulation) is characterized by its ability to induce photobiological processes in cells. Exact action spectra are needed for determination of photoacceptors as well as for further investigations into cellular mechanisms of phototherapy.

METHODS: Seven action spectra for the stimulation of DNA and RNA synthesis rate and cell adhesion to glass matrix are analyzed by curve fitting, followed by deconvolusion with Lorentzian fitting. Exact parameters of peak positions and bandwidths are presented.

RESULTS: The peak positions are between 613.5 and 623.5 nm (in one spectrum, at 606 nm), in the red maximum. The far-red maximum has exact peak positions between 667.5 and 683.7 nm in different spectra. Two near infrared maxima have peak positions in the range 750.7-772.3 nm and 812.5-846.0 nm, respectively.

CONCLUSIONS: In the wavelength range important for phototherapy (600-860 nm), there are four “active” regions, but peak positions are not exactly the same for all spectra.

Photomed Laser Surg. 2005 Jun;23(3):268-72.

Effects of 670-nm phototherapy on development.

Yeager RL, Franzosa JA, Millsap DS, Angell-Yeager JL, Heise SS, Wakhungu P, Lim J, Whelan HT, Eells JT, Henshel DS.

School of Public and Environmental Affairs, Indiana University, Bloomington, IN 47405, USA. rlyeager@indiana.edu

OBJECTIVE: The objective of the present study was to assess the survival and hatching success of chickens (Gallus gallus) exposed in ovo to far-red (670-nm) LED therapy.

BACKGROUND DATA: Photobiomodulation by light in the red to near-infrared range (630-1000 nm) using low-energy lasers or light-emitting diode (LED) arrays has been shown to accelerate wound healing and improve recovery from ischemic injury. The mechanism of photobiomodulation at the cellular level has been ascribed to the activation of mitochondrial respiratory chain components resulting in initiation of a signaling cascade that promotes cellular proliferation and cytoprotecton.

MATERIALS AND METHODS: Fertile chicken eggs were treated once per day from embryonic days 0-20 with 670-nm LED light at a fluence of 4 J/cm2. In ovo survival and death were monitored by daily candling (after Day 4).

RESULTS: We observed a substantial decrease in overall and third-week mortality rates in the light-treated chickens. Overall, there was approximately a 41.5% decrease in mortality rate in the light-treated chickens (NL: 20%; L: 11.8%). During the third week of development, there was a 68.8% decrease in the mortality rate in light-treated chickens (NL: 20%; L: 6.25%). In addition, body weight, crown-rump length, and liver weight increased as a result of the 670-nm phototherapy. Light-treated chickens pipped (broke shell) earlier and had a shorter duration between pip and hatch.

CONCLUSION: These results indicate that 670-nm phototherapy by itself does not adversely affect developing embryos and may improve the hatching survival rate.

Lasers Surg Med. 2005 Apr;36(4):307-14.

Cellular effects of low power laser therapy can be mediated by nitric oxide.

Karu TI, Pyatibrat LV, Afanasyeva NI.

Institute of Laser and Information Technologies of the Russian Academy of Sciences, 142190 Troitsk, Moscow, Russia. tkaru@isan.troitsk.ru

Abstract

BACKGROUND AND OBJECTIVES: The objective of this study was to investigate the possibility of involvement of nitric oxide (NO) into the irradiation-induced increase of cell attachment. These experiments were performed with a view to exploring the cellular mechanisms of low-power laser therapy.

STUDY DESIGN/MATERIALS AND METHODS: A suspension of HeLa cells was irradiated with a monochromatic visible-to-near infrared radiation (600-860 nm, 52 J/m2) or with a diode laser (820 nm, 8-120 J/m2) and the number of cells attached to a glass matrix was counted after 30 minute incubation at 37 degrees C. The NO donors sodium nitroprusside (SNP), glyceryl trinitrate (GTN), or sodium nitrite (NaNO2) in the concentration range 5 x 10(-9)-5 x 10(-4)M were added to the cellular suspension before or after irradiation. The action spectra and the concentration and fluence dependencies obtained were compared and analyzed.

RESULTS: The well-structured action spectrum for the increase of the adhesion of the cells, with maxima at 619, 657, 675, 740, 760, and 820 nm, points to the existence of a photoacceptor responsible for the enhancement of this property (supposedly cytochrome c oxidase, the terminal respiratory chain enzyme), as well as signaling pathways between the cell mitochondria, plasma membrane, and nucleus. Treating the cellular suspension with SNP (5 x 10(-5)M) before irradiation significantly modifies the action spectrum for the enhancement of the cell attachment property (band maxima at 642, 685, 700, 742, 842, and 856 nm). The action of SNP, GTN, and NaNO2 added before or after irradiation depends on their concentration and radiation fluence.

CONCLUSIONS: The NO donors added to the cellular suspension before irradiation eliminate the radiation-induced increase in the number of cells attached to the glass matrix, supposedly by way of binding NO to cytochrome c oxidase. NO added to the suspension after irradiation can also inhibit the light-induced signal downstream. Both effects of NO depend on the concentration of the NO donors added. These results indicate that NO can control the irradiation-activated reactions that increase the attachment of cells.

J Biol Chem. 2005 Feb 11;280(6):4761-71. Epub 2004 Nov 22.

Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase.

Wong-Riley MT, Liang HL, Eells JT, Chance B, Henry MM, Buchmann E, Kane M, Whelan HT.

Department of Cell Biology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. mwr@mcw.edu

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 microm KCN. It significantly reduced neuronal cell death induced by 300 microm 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 microm KCN but not at higher concentrations of KCN tested. Pretreatment with LED enhanced efficacy of LED during exposure to 10 or 100 microm 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.

Mitochondrion. 2004 Sep;4(5-6):559-67.

Mitochondrial signal transduction in accelerated wound and retinal healing by near-infrared light therapy.

Eells JT, Wong-Riley MT, VerHoeve J, Henry M, Buchman EV, Kane MP, Gould LJ, Das R, Jett M, Hodgson BD, Margolis D, Whelan HT.

Department of Health Sciences, College of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA. jeells@uwm.edu

Photobiomodulation by light in the red to near infrared range (630-1000 nm) using low energy lasers or light-emitting diode (LED) arrays has been shown to accelerate wound healing, improve recovery from ischemic injury in the heart and attenuate degeneration in the injured optic nerve. Recent evidence indicates that the therapeutic effects of red to near infrared light result, in part, from intracellular signaling mechanisms triggered by the interaction of NIR light with the mitochondrial photoacceptor molecule cytochrome c oxidase. We have demonstrated that NIR-LED photo-irradiation increases the production of cytochrome oxidase in cultured primary neurons and reverses the reduction of cytochrome oxidase activity produced by metabolic inhibitors. We have also shown that NIR-LED treatment prevents the development of oral mucositis in pediatric bone marrow transplant patients. Photobiomodulation improves wound healing in genetically diabetic mice by upregulating genes important in the promotion of wound healing. More recent studies have provided evidence for the therapeutic benefit of NIR-LED treatment in the survival and functional recovery of the retina and optic nerve in vivo after acute injury by the mitochondrial toxin, formic acid generated in the course of methanol intoxication. Gene discovery studies conducted using microarray technology documented a significant upregulation of gene expression in pathways involved in mitochondrial energy production and antioxidant cellular protection. These findings provide a link between the actions of red to near infrared light on mitochondrial oxidative metabolism in vitro and cell injury in vivo. Based on these findings and the strong evidence that mitochondrial dysfunction is involved in the pathogenesis of numerous diseases processes, we propose that NIR-LED photobiomodulation represents an innovative and non-invasive therapeutic approach for the treatment of tissue injury and disease processes in which mitochondrial dysfunction is postulated to play a role including diabetic retinopathy, age-related macular degeneration, Leber’s hereditary optic neuropathy and Parkinson’s disease.

Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3439-44. Epub 2003 Mar 7.

Therapeutic photobiomodulation for methanol-induced retinal toxicity.

Eells JT, Henry MM, Summerfelt P, Wong-Riley MT, Buchmann EV, Kane M, Whelan NT, Whelan HT.

Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. jeells@uwm.edu

Methanol intoxication produces toxic injury to the retina and optic nerve, resulting in blindness. The toxic metabolite in methanol intoxication is formic acid, a mitochondrial toxin known to inhibit the essential mitochondrial enzyme, cytochrome oxidase. Photobiomodulation by red to near-IR radiation has been demonstrated to enhance mitochondrial activity and promote cell survival in vitro by stimulation of cytochrome oxidase activity. The present studies were undertaken to test the hypothesis that exposure to monochromatic red radiation from light-emitting diode (LED) arrays would protect the retina against the toxic actions of methanol-derived formic acid in a rodent model of methanol toxicity. Using the electroretinogram as a sensitive indicator of retinal function, we demonstrated that three brief (2 min, 24 s) 670-nm LED treatments (4 J/cm(2)), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. Our studies document a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats. We further show that LED treatment protected the retina from the histopathologic changes induced by methanol-derived formate. These findings provide a link between the actions of monochromatic red to near-IR light on mitochondrial oxidative metabolism in vitro and retinoprotection in vivo. They also suggest that photobiomodulation may enhance recovery from retinal injury and other ocular diseases in which mitochondrial dysfunction is postulated to play a role.

Neuroreport. 2001 Oct 8;12(14):3033-7.

Light-emitting diode treatment reverses the effect of TTX on cytochrome oxidase in neurons.

Wong-Riley MT, Bai X, Buchmann E, Whelan HT.

Department of Cell Biology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.

Light close to and in the near-infrared range has documented benefits for promoting wound healing in human and animals. However, mechanisms of its action on cells are poorly understood. We hypothesized that light treatment with a light-emitting diode array at 670 nm (LED) is therapeutic in stimulating cellular events involving increases in cytochrome oxidase activity. LED was administered to cultured primary neurons whose voltage-dependent sodium channels were blocked by tetrodotoxin. The down-regulation of cytochrome oxidase activity by TTX was reverted to control levels by LED. LED alone also up-regulated enzyme activity. Thus, the results are consistent with our hypothesis that LED has a stimulating effect on cytochrome oxidase in neurons, even when they have been functionally silenced by TTX.

Int J Radiat Biol. 2000 Jun;76(6):863-70.

Specific helium-neon laser sensitivity of the purified cytochrome c oxidase.

Pastore D, Greco M, Passarella S.

Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Campobasso, Italy.

Abstract

PURPOSE: In order to gain some insight into the mechanism of interaction between Helium-Neon (He-Ne) laser light and mitochondrial cytochromes, the sensitivity of cytochrome electron transfer activity to He-Ne laser was tested.

MATERIALS AND METHODS: Irradiation of solutions containing either purified cytochromes or dissolved rat liver mitochondria was carried out (wavelength 632.8 nm, fluence rate 10 mW cm(-2), fluence 2 J cm(-2)); the irradiation conditions were the ones able to affect cytochrome c oxidase (COX) activity in mitochondria (Pastore et al., 1994).

RESULTS: Cytochrome c oxidation catalysed by COX was affected by He-Ne laser irradiation of the purified enzyme. This result was obtained from measurements of the pseudo-first-order kinetic constant and from determinations of the turnover number of the enzyme, performed at different cytochrome c/COX ratios. Consistently, the kinetic parameters of COX changed. On the contrary, no alteration in the rate of electron transfer catalysed by either cytochrome c or bc1 complex was found.

CONCLUSIONS: This study shows that purified COX is a specific target of He-Ne laser light; therefore, COX may be considered to be a mitochondrial photo-acceptor.

J Photochem Photobiol B. 1999 Mar;49(1):1-17.

Primary and secondary mechanisms of action of visible to near-IR radiation on cells.

Karu T.

Laser Technology Research Center of Russian Academy of Sciences, Troitsk, Moscow Region, Russia. kara@isan.troitsk.ru

Cytochrome c oxidase is discussed as a possible photoacceptor when cells are irradiated with monochromatic red to near-IR radiation. Four primary action mechanisms are reviewed: changes in the redox properties of the respiratory chain components following photoexcitation of their electronic states, generation of singlet oxygen, localized transient heating of absorbing chromophores, and increased superoxide anion production with subsequent increase in concentration of the product of its dismutation, H2O2. A cascade of reactions connected with alteration in cellular homeostasis parameters (pHi, [Cai], cAMP, Eh, [ATP] and some others) is considered as a photosignal transduction and amplification chain in a cell (secondary mechanisms).