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

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 (Δ[CCO]) and oxygenated hemoglobin concentration (Δ[HbO]) on the treated site as the laser energy dose accumulated over time. A strong linear interplay between Δ[CCO] and Δ[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.

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 treatment; *indicates significant differences in respective concentrations between LLLT and placebo treatment (0.01 < p < 0.05, paired t-test). **indicates significant differences in respective concentrations between LLLT and placebo treatment (p < 0.01, paired t-test).

Figure 2. The relationship of concentration changes between CCO vs. HbO or Hb during LLLT and placebo experiment (mean ± SE, N = 11).

The horizontal error bars represent variability of CCO and the vertical error bars represent variability of HbO or Hb. The correlation coefficient of the fitted line is r = 0.92 with a p value of 0.001.

Figure 3. Schematic diagram of the experimental setup, including the broadband NIRS system.

This bb-NIRS consisted of a tungsten halogen lamp as light source and a miniature back-thinned CCD spectrometer as detector. 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 from the tungsten halogen lamp to the tissues.

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 bundles with a separation of 1.5 cm. One bundle (in red) was connected to the tungsten halogen lamp and the other (in blue color) to the spectrometer. The LLLT/placebo treatments were administered on two sides of the middle section alternatively (pink circles).

Figure 5. Paradigm of the LLLT/placebo treatment and interleaved bb-NIRS data acquisition.

Each treatment session consisted of eight one-minute treatment cycles, 55-s laser on and 5-s laser off per cycle. The bb-NIRS data acquisition was initiated two minutes before the first treatment session and ceased five minutes after the treatment session.

Figure 6. A flow chart describing detailed procedures of our multiple linear regression analysis to optimally determine LLLT-induced concentration changes in three chromophores.