Up-regulation of cerebral cytochrome-c-oxidase and hemodynamics by transcranial infrared laser stimulation: A broadband near-infrared spectroscopy study

Abstract

Transcranial infrared laser stimulation (TILS) is a noninvasive form of brain photobiomulation. Cytochrome-c-oxidase (CCO), the terminal enzyme in the mitochondrial electron transport chain, is hypothesized to be the primary intracellular photoacceptor. We hypothesized that TILS up-regulates cerebral CCO and causes hemodynamic changes. We delivered 1064-nm laser stimulation to the forehead of healthy participants ( n = 11), while broadband near-infrared spectroscopy was utilized to acquire light reflectance from the TILS-treated cortical region before, during, and after TILS. Placebo experiments were also performed for accurate comparison. Time course of spectroscopic readings were analyzed and fitted to the modified Beer-Lambert law. With respect to the placebo readings, we observed (1) significant increases in cerebral concentrations of oxidized CCO (Δ[CCO]; >0.08 µM; p < 0.01), oxygenated hemoglobin (Δ[HbO]; >0.8 µM; p < 0.01), and total hemoglobin (Δ[HbT]; >0.5 µM; p < 0.01) during and after TILS, and (2) linear interplays between Δ[CCO] versus Δ[HbO] and between Δ[CCO] versus Δ[HbT]. Ratios of Δ[CCO]/Δ[HbO] and Δ[CCO]/Δ[HbT] were introduced as TILS-induced metabolic-hemodynamic coupling indices to quantify the coupling strength between TILS-enhanced cerebral metabolism and blood oxygen supply. This study provides the first demonstration that TILS causes up-regulation of oxidized CCO in the human brain, and contributes important insight into the physiological mechanisms.

Keywords: Cerebral hemodynamics; energy metabolism; mitochondria; near-infrared spectroscopy; neuroprotection.

Figure 1.

Schematic diagram of the experimental setup, including a bb-NIRS spectroscopic system. This bb-NIRS unit consisted of a tungsten halogen lamp as the light source and a miniature high-sensitivity CCD spectrometer as the detector for this study. TILS was administered underneath the “I”-shaped probe holder. The narrow, middle section of the holder was ∼8 mm in width. A laptop computer was used to acquire, display and save the data from the spectrometer. The shutter controlled the on and off function for the white light from the tungsten-halogen lamp to the subject’s forehead. A pair of protection goggles was worn during the whole experimental procedure.

Figure 2.

Paradigm of the TILS/placebo treatment and interleaved bb-NIRS data acquisition. Each treatment session consisted of eight 1-min treatment cycles: 55-s laser on and 5-s laser off per cycle. During the 5-s laser-off periods, the bb-NIRS system (both the light source and detector) was switched on for bb-NIRS data acquisition. The same data acquisition format was followed for baseline and recovery sessions.

Figure 3.

Subject-averaged time courses of TILS/placebo-induced cerebral changes of (a) [HbO], (b) [HHb], (c) [HbT], (d) [HbD], and (e) [CCO] (all in µM) recorded from human foreheads in vivo (mean ± SE, n = 11). Time zero (t = 0) is the onset of TILS. In each subplot, the shaded region indicates the period of TILS/placebo treatment. “*” indicates significant differences (0.01 < p < 0.05, two sample t-test) in respective concentrations between TILS and placebo treatment. “**” indicates significant differences (p < 0.01, two sample t-test) in respective concentrations between TILS and placebo treatment.

Figure 4.

(a) Relationships between subject-averaged Δ[CCO] vs Δ[HbO] and Δ[CCO] vs Δ[HHb] that resulted from TILS and placebo treatment (mean ± SE, N = 11). Solid red diamonds display the relationship of Δ[CCO] vs Δ[HbO]; solid blue dots display the relationship of Δ[CCO] vs Δ[HHb]. Both red open diamonds and blue open squares represent placebo-treated Δ[CCO] vs Δ[HbO] and Δ[CCO] vs Δ[HHb], respectively. (b) Relationships between subject-averaged Δ[CCO] vs Δ[HbT] and Δ[CCO] vs Δ[HbD] caused by TILS and placebo treatment. Solid purple circles display the relationship of Δ[CCO] vs Δ[HbT]; solid black triangles display the relationship of Δ[CCO] vs Δ[HbD]. Both red open circles and open triangles represent placebo-treated Δ[CCO] vs Δ[HbO] and Δ[CCO] vs Δ[HHb], respectively. All error bars represent standard errors of means from respective chromophore concentrations.

Figure 5.

Ratios of (a) Δ[CCO]/Δ[HbO], (c) Δ[CCO]/Δ[HbT], and (e) Δ[CCO]/Δ[HbD] during and after infrared laser stimulation on the right forehead of 11 human subjects (red symbols) and on the right forearm of another group of 11 human subjects (blue symbols). Time-averaged ratios of (b) Δ[CCO]/Δ[HbO], (d) Δ[CCO]/Δ[HbT], and (f) Δ[CCO]/Δ[HbD] with corresponding standard deviations in both forearm and forehead stimulation cases. “**” marks significant difference (p < 0.01, two sample t-test) of Δ[CCO]/Δ[HbO] or Δ[CCO]/Δ[HbT] between the stimulated cases. “*” marks significant difference (p < 0.05, two sample t-test) of Δ[CCO]/Δ[HbD] between the stimulated cases.

Figure 6.

(a) Model of the photobiochemical mechanism of action of infrared light on the measured cytochrome c oxidase oxidation (CCO oxidized) and hemoglobin oxygenation (HbO₂). While some of the initial steps may occur at the same time, the proposed biochemical processes are numbered sequentially to better explain the observed increased concentration of oxidized CCO during 1064-nm laser stimulation. See text for detailed explanation. (b) The right most panel shows a pyramidal neuron of the cerebral cortex, which is modified from a figure in Huettel et al. The middle dashed rectangle is a zoomed section of apical dendrite, containing an abundant amount of mitochondria, with which CCO can be photo-stimulated and oxidized by TILS. This photobiomodulation consequently drives increases of HbO, HbT, and HbD. (c) A flow chart to show the conventional neuro-vascular coupling by the black-colored notations and our newly-defined TILS-induced metabolic-hemodynamic coupling by the red-colored notations. The blue-colored notations represent common endpoints of both mechanisms on the cerebral circulation.