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 cm 2 area in the hind limb at 670 nm (50 mW/cm 2) 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.
The proper physiological function of tissues relies on the timely and appropriate delivery of blood flow. Disruption or derangement of blood flow, either through physical obstruction or impaired responses to cellular agonists, results in ischemia and end organ damage. Among numerous vasodilators the nitric oxide (NO) family represents an effective means to return blood flow to ischemic tissues (Adams et al., 1997; Homer and Wanstall, 2002; Liu et al., 2016). However, the pharmacological use of NO donors is limited by tolerance or medication side effects (Miller et al., 2000; Lundberg et al., 2009; Mohler et al., 2014). Moreover, endogenous production of NO is attenuated in cardiovascular diseases by reduced expression and uncoupling of its associated enzyme endothelial nitric oxide synthase (eNOS). Alternative methods for increasing endogenous NO (Lohr et al., 2009; Shiva and Gladwin, 2009; Keszler et al., 2018; Wajih et al., 2019; Wajih et al., 2021) have identified nitrite, nitrosyl and S-nitrosothiol (RSNO) containing proteins as potential sources. These compounds do not display pharmacological tolerance, nor do they require optimal intracellular oxygen and pH to function. As an alternative, photo relaxation of conduit vessels in the red or near infrared region of the electromagnetic spectrum has gained growing recognition as a non-invasive targeted modality to increase NO supply (Karu et al., 2005; Poyton and Ball, 2011; Ieda et al., 2020; Colombo et al., 2021).
Photobiomodulation (PMB) or low-level laser (light) therapy (LLLT), is the irradiation of tissues with laser or LED in the visible and near infrared range of the electromagnetic spectrum. The cellular mechanisms of photobiomodulation are well described, e. g. proliferation, migration, cytoprotection, inflammation, which contribute to accelerated tissue repair such as wound healing (Freitas and Hamblin, 2016). Irradiation affects a variety of cellular functions with implication of multiple regulatory mechanisms (Karu, 1999). One potential hypothesis for light’s action attributes a central role of complex IV (cytochrome c oxidase, Cco) of the electron transport chain (Karu, 1999; Wong-Riley et al., 2005; Karu T., 2010; Hamblin, 2016), however, numerous other reaction routes may contribute to the general network. Some pathways involve NO which is canonically produced via the activity of its synthase enzymes but can also enter the cells from outside by diffusion, or from nitrate and nitrite containing nutrients. The latter can be reduced in many ways (Machha and Schechter, 2011), and specifically in the mitochondrion by the reductase activity of Cco (Poyton and Ball, 2011) or the ubiquionone cycle (Kozlov et al., 1999).
670 nm light facilitates the availability of these NO precursors from their stores (Wong-Riley et al., 2005; Machha and Schechter, 2011), and induces NO release outside of the mitochondria related mechanisms from nitrosylated heme (Lohr et al., 2009; Shiva and Gladwin, 2009), non heme dinitrosyl iron complexes (DNIC) or S-nitrosothiols (RSNO) (Keszler et al., 2018; Wajih et al., 2019; Wajih et al., 2021). Red light synergistically enhances the vasodilatory effect of nitrite (Lohr et al., 2009; Wajih et al., 2019; Wajih et al., 2021) and is capable to liberate NO from nitrosyl and nitroso derivatives (Lohr et al., 2009; Keszler et al., 2018). N-nitroso compounds (RNNO) may also be a putative target of photolytic cleavage to NO, however, mainly at shorter wavelengths, and with a minor relaxing effect (Rodriguez et al., 2003).
Red light treatments reduce the damage observed in traumatic brain injury and stroke models (Hamblin, 2016), as well as cardiac ischemia-reperfusion injury through NO mechanisms (Lohr et al., 2009; Shiva and Gladwin, 2009). Small case studies using red light as a therapy for diabetic wounds (Desmet et al., 2006) and chronic venous ulcers (Caetano et al., 2009) suggest clinical relevance.
In earlier studies (Lohr et al., 2009; Keszler et al., 2017; Keszler et al., 2018; Keszler et al., 2019; Weihrauch et al., 2021) we recognized that 670 nm wavelength is optimal to achieve ex vivo dilation of dissected blood vessel and the light effect is not hindered by temperature interference. The vasodilation occurs via activation of the NO pathway. Investigation of the underlying mechanism revealed a transferable species secreted from the endothelium which is stable and active ex vivo for at least for 30 min, and contains NO moiety, and most probably also free NO. The secretion of the dilatory species is under the control of red light.
Here we examined the stability of the endothelium derived NO precursor in an in vivo mouse model by detecting blood flow and measuring the levels of the vasodilatory species in plasma and tissue samples collected at the end point. We optimized the conditions by which 670 nm light would maximize blood flow and determined the stability of the vasoactive NO precursor in plasma and muscle tissue under optimal conditions. Then, in order to mimic the clinical conditions of peripheral artery disease (PAD), we placed an ameroid constrictor, a device containing hygroscopic material to partially occlude the vessel, on the proximal femoral artery of mice. Then measured the arteriolar blood flow reduction and its restoration by red light treatment.
All used chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated.
The 670 nm LED lamps with power supply were purchased from Quantum Devices Inc. (Barneveld, WI). The power output was measured with X97 Optometer (Gigahertz Optics, Turkenfeld, Germany). The used parameters are summarized in Table 1.
Photobiomodulation parameters.
C57BL/6 mice (26.4 ± 0.4 g, n = 10) were purchased from Jackson Laboratories. Power analysis for ANOVA was used to determine the number of animals required. The overall mean difference between the ameroid and control groups was about 170 and the standard deviation was about 210. The correlation coefficient was 0.63. The post-hoc power using a paired t-test with a one-sided 0.05 significance level based on these parameters obtained from the data is about 86.4% to detect a difference between the ameroid and control groups. The other figures are for the exploratory analysis. All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin. Furthermore, all procedures and protocols conformed to the Guiding Principles in the Care and Use of Animals of the American Physiologic Society and were in accordance with the Guide for the Care and Use of Laboratory Animals. Protocol number is 2,969. For laser doppler measurement mice were anesthetized with 1.25% isoflurane-O 2. A heat mat was used to control the animal’s temperature during LDI. Since a black background is necessary for proper LDI imaging, the heat mat was placed underneath the imaging mat. Rectal temperature was maintained at 37.0 ± 0.5°C for the duration of the surgery and during LD. The hair on the hindlimb and thigh region of the posterior side of the mouse was removed. Hindlimb perfusion was assessed noninvasively in the plantar foot (index of the overall leg perfusion) before and immediately after red light treatment (670 nm) by Scanning Laser-Doppler (model LDI2-IR; Moor Instruments, Wilmington, DE). For the optimization, the left paw received no light and served as control for each animal, a 3 cm 2 area of the right paw was treated with 0–100 mW/cm 2 of 670 nm light for 10 min. Doppler perfusion of the plantar foot was assessed within anatomically defined regions of interest, consisting of the hind paw margins. The change in Doppler Intensity Units (DIU) pre- and post-red-light treatment was first determined in each paw. When examining the stability of the NO precursor vasodil
