In Vivo Characterization of a Red Light-Activated Vasodilation: A Photobiomodulation Study

Abstract

Nitric oxide dependent vasodilation is an effective mechanism for restoring blood flow to ischemic tissues. Previously, we established an ex vivo murine model whereby red light (670 nm) facilitates vasodilation via an endothelium derived vasoactive species which contains a functional group that can be reduced to nitric oxide. In the present study we investigated this vasodilator in vivo by measuring blood flow with Laser Doppler Perfusion imaging in mice. The vasodilatory nitric oxide precursor was analyzed in plasma and muscle with triiodide-dependent chemiluminescence. First, a 5-10 min irradiation of a 3 cm² area in the hind limb at 670 nm (50 mW/cm²) produced optimal vasodilation. The nitric oxide precursor in the irradiated quadriceps tissue decreased significantly from 123 ± 18 pmol/g tissue by both intensity and duration of light treatment to an average of 90 ± 17 pmol/g tissue, while stayed steady (137 ± 21 pmol/g tissue) in unexposed control hindlimb. Second, the blood flow remained elevated 30 min after termination of the light exposure. The nitric oxide precursor content significantly increased by 50% by irradiation then depleted in plasma, while remained stable in the hindlimb muscle. Third, to mimic human peripheral artery disease, an ameroid constrictor was inserted on the proximal femoral artery of mice and caused a significant reduction of flow. Repeated light treatment for 14 days achieved steady and significant increase of perfusion in the constricted limb. Our results strongly support 670 nm light can regulate dilation of conduit vessel by releasing a vasoactive nitric oxide precursor species and may offer a simple home-based therapy in the future to individuals with impaired blood flow in the leg.

Keywords: endothelium; nitric oxide; photobiomodulation; red light therapy; vasodilation.

FIGURE 1

Optimization of vasodilatory effect of red light. 670 nm light was applied at 25, 50, 100 mW/cm² for 5–15 min in hind limb of C57BI/6 mice and blood flow was measured with Laser Doppler Imaging. The flow was measured as a difference between the irradiated limb and the other (control) limb of each animal. (A): Representative image. (B): Quantification of paw perfusion. Mean ± SD is denoted. The Laser Doppler Intensity in irradiated group was significantly greater than that in the control group (p < 0.0001). In Friedman’s two-way nonparametric ANOVA, a statistical significance was detected for the exposure duration (p = 0.0102), but not for power density (p = 0.177).

FIGURE 2

Release of NO precursor species at varying power of red light. Ozone-based chemiluminescence analysis was carried out with triiodide-dependent chemiluminescence in plasma (A), Quadriceps muscle treated with light (B) or control limb (C). Mean ± SD is denoted. (A): No statistical significance was detected between the irradiated and control group (p = 0.6108). (B): The difference is significant between the irradiated and control groups (p = 0.0280). No statistical significance was detected for either power density or exposure durations (p = 0.7932 and 0.2902, respectively). (C): No statistical significance was detected between the irradiated and control group (P-value: 0.6323).

FIGURE 3

Stability of red light-dependent vasodilator. 670 nm light was applied at 50 mW/cm² for 10 min on entire body of C57BI/6 mice with and the vasodilation was assessed at 0, 5, 10, and 30 min after finishing the irradiation. (A): Blood flow measured with Laser Doppler Imaging. (B, C): The NO-derived vasodilator was measured with ozone-based chemiluminescence in plasma (B), and right quadriceps tissue (C). Mean ± SD is denoted. (A): The difference is significant between each light treatment group compared to the control group (The adjusted P-values by the step-down Bonferroni procedure are 0.0013, 0.0052, 0.0011, and 0.0711, respectively). (B): The overall P-value was 0.0249. Dunnett’s multiple comparison test suggested the “Light” group demonstrated a significant difference compared to the control group (adjusted p = 0.0469). The other three light treatments groups did not demonstrate a significant difference. (C): There was no significant difference between the irradiated and control groups (overall p = 0.987).

FIGURE 4

Stability of red light-dependent vasodilator. 670 nm light was applied at 50 mW/cm² for 10 min on right limb of C57BI/6 mice with blood flow was measured with Laser Doppler Imaging. Difference between the irradiated and non-irradiated limbs. Vasodilation was assessed at 0, 5, 10, and 30 min after finishing the irradiation. Mean ± SD is denoted. There was no significant difference in the “No light control” groups (the adjusted P-value by the step-down Bonferroni procedure is 0.3989). In the “Light” group for each time point, there was a significant difference compared to control (adjusted p = 0.0189, 0.0189, 0.0330, and 0.0017, respectively).

FIGURE 5

Ameroid constrictor to mimic hindlimb ischemia. (A, C): A representative 0.25 mm internal diameter ameroid constrictor designed, constructed, and implanted on the proximal femoral artery by our laboratory. (B): Laser Doppler Images of C57Bl/6 mice treated with an ameroid in the femoral artery and the contralateral control. (D): Blood flow measured with Laser Doppler Imaging. Mean ± SD is denoted. The adjusted P-values by the step-down Bonferroni procedure between control and ameroid are p = 1, 0.162, 0.00178, 0,00101, 2.37 × 10^-8, 0.932, 0.000202, 0.00868, and 0.289, respectively.

FIGURE 6

Light effect on ameroid hindlimb ischemia. Doppler perfusion of the plantar foot was assessed pre and post-surgical implantation of the ameroid and for 14 days post-surgery immediately before and after light treatment. (A–C) Blood Flow measured by Laser Doppler Imaging before and after 670 nm light on ameroid paw: Ameroid paw (A), Control paw (B), and comparison of both paws after irradiation (C). Mean ± SD is denoted. The adjusted P-values by the step-down Bonferroni procedure between control and ameroid are p = 0.165, 3.70 × 10^-15, 1.25 × 10^-9, 6.30 × 10^-14, 1.99 × 10^-5, 2.28 × 10^-4, 9.24 × 10^-18, and 2.57 × 10^-4, respectively (A), 0.222, 0.146, 0.0284, 0.421, 0.406, 1, 1, and 0.406, respectively (B) or control and ameroid 1, 1, 0.343, 1, 0.289, 0.107, 0.136, and 0.0143(C), respectively.