Application of Photobiomodulation Therapy by LED Using a Combined Red and Near-infrared Light Spectrum Improves the Vascular Function of the Radial Artery in Vitro
Phase: NA
Conditions: Vascular Dysfunction
Sponsor: University of Sao Paulo

Official Title of the study  
  
Application of photobiomodulation therapy by LED using a combined red and 
nearinfrared light spectrum improves the vascular function of the radial artery in 
vitro.   
 
Protocol no. 4.829.712, approved on April 22, 2023 
 
Authors: André Timóteo Sapalo, PhD1,2,5; Carlos Henrique Miranda, PhD2; Rinaldo  
Roberto de Jesus Guirro PhD1; Elaine Caldeira de Oliveira Guirro PhD3; Carlos Eduardo   
Girasol PhD2; Gisele Moceline Junkoski PhD3   
1HCRP-USP Echocardiography Laboratory, Department of Clinical Medicine, 
FMRPUSP, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto -SP, 
Brazi   
2Division of Emergency Medicine, Department of Internal Medicine, Vascular Biology 
Laboratory, School of Medicine, Ribeirão Preto, University of São Paulo, Brazil.   
 
 
 
                                                 
1 Postgraduate Program in Rehabilitation and Functional Performance, Department of 
Health Science, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto 
– SP, Brazil.   
2 Postgraduate Program in Physics Applied to Medicine and Biology, University of São 
Paulo (USP), Ribeirão Preto, Brazil.   
3 Department of Cardiac Surgery, Hospital das Clínicas, University of São Paulo.   
   

Abstract   
LED photobiomodulation therapy (PBMT) is widely recognized for its beneficial effects 
in several clinical conditions; however, its potential in modulating vascular function is 
not yet fully established. Objective: This study aimed to evaluate the application of 
PBMT by LED (light-emittion diode) using a combined red and near-infrared light 
spectrum, with a focus on improving the vascular function of the radial artery in vitro. 
Methodology: A total of 30 radial artery segments were studied and divided into three 
groups of 10 segments: the PBMT LED group combined with red and infrared light (R + 
IR), the red light (R) group, and the infrared light (IR) group. Prior to light irradiation at  
wavelengths of 650 nm and 940 nm, vascular function in these segments was 
pharmacologically inhibited using L-NAME. The drugs phenylephrine, acetylcholine, 
and sodium nitroprusside were used to determine if irradiation could restore the inhibited 
vascular functions. Superoxide anion levels, an important reactive oxygen specie (ROS), 
were measured using a chemiluminescence assay with lucigenin. The bioavailability of 
nitric oxide was evaluated using a Griess Reagent Kit assay, while nitric oxide production  
was assessed through the DAF fluorescence assay. The vascular inflammation was 
determined by the Intercellular Adhesion Molecule 1 (ICAM-1) expression.    
   
Key words: Photobiomodulation; light-emitting diode; vascular function; radial artery 
Introduction   
LED photobiomodulation therapy (PBMT) is widely recognized for its beneficial 
effects in various clinical conditions. This technique is used as a therapeutic resource for 
a variety of pathologies in various fields, including musculoskeletal disorders1, 
dermatological conditions2, dental issues3, and even mood disorders4, with increasingly 
promising results.   
When applied with appropriate parameters, PBMT has the potential to induce 
fundamental biological changes in tissue via the absorption of light photons, stimulating 
or inhibiting various processes as needed5. To achieve these outcomes, PBMT can be 
administered through different methods, either directly in contact with the target area or 
systematically via the vascular system, which may involve transdermal or intravascular  
application6.   
PBMT has found extensive application, particularly in the treatment of  

cardiovascular diseases7. Initially, the technique involved the application of laser light 
through an intravenous catheter, typically inserted into the radial artery. This approach, 
known as intravascular laser irradiation of blood (ILIB)8, was first reported by Russian 
researchers (PMID: 3191241), who attributed the therapeutic effects to the normalization 
of blood lipid levels, as well as the reduction of platelet aggregation and clot formation. 
Furthermore, it is suggested that the technique may activate the immune system, 
involving dendritic cells, macrophages, and lymphocytes9.   
For a long time, the evolution of this technique stagnated due to the limitations of 
its intravenous application. However, in recent years, modifications have been made to 
facilitate its use, and systemic PBMT has begun to be applied transdermally, such as on 
the radial artery. This approach, known as vascular photobiomodulation (VPBM), has 
been indicated for clinical use10. The ease of application has led to an expansion of the 
technology's use in clinical settings; however, the scientific validation of its effects 
remains unclear in light of available clinical reports.   
In recent years, photobiomodulation mediated by light-emitting diodes (LEDs) has 
gained significant prominence and clinical focus as a more viable and cost-effective 
therapeutic option. Compared with laser devices, LEDs offer several advantages, 
including smaller size, lighter weight, lower cost, and ease operation11,12. Although some  
studies have demonstrated that LED irradiation can promote improvements in 
microcirculation and enhance the healing of diabetic ulcers in clinical settings13,14, the 
dose-response relationship and underlying mechanisms of LED-mediated PBMT remain 
unclear. Several parameters are crucial for PBMT, including wavelength, light irradiance, 
energy, treatment duration, and frequency. Red or near-infrared light (600–1000 nm) is 
generally considered the optimal range for enhancing vascular function15. The present 
study aimed to assess the effects of LED photobiomodulation therapy on vascular 
function in vitro using human radial arteries harvested during coronary artery bypass 
grafting.   
Materials and Methods Ethical aspects   
This study was approved by the Human Research Ethics Committee of the 
Hospital das Clínicas, FMRP/USP (protocol no. 4.829.712, approved on April 22, 
2023). The research was conducted in the Vascular Physiology Laboratory, Department 

of Physiology, University of São Paulo. Surgical patients who consented to donate a 
segment of their radial artery were fully informed about the study objectives and the 
intended use of the donated tissue. Written informed consent was obtained from all  
participants.  
Removal of radial arteries   
Radial arteries (RAs) were harvested during coronary artery bypass grafting 
(CABG) by surgeons at Hospital das Clínicas – FMRP/USP – Brazil. Harvesting the 
radial artery is a standard procedure and is the most commonly used artery for coronary 
artery bypass grafting due to its favorable characteristics. All surgeries were performed 
by the same surgical team using cardiopulmonary bypass and cardioplegic arrest. The 
radial artery was primarily used to provide secondary target vessels, mainly the obtuse 
marginal branches or posterior descending arteries, in 30 patients. The radial artery was 
selected for this purpose due to its ability to yield a sufficiently long segment, making it 
suitable for use as a coronary graft, with the remaining tissue being used for the present 
study.   
Before radial artery extraction, all patients underwent Doppler vascular 
assessments to evaluate arterial function. An ultrasound system equipped with a 13-6 
MHz linear transducer (HFL38x/13-6 MHz Transducer, FUJIFILM Sonosite, Inc., 
Bothell, WA, USA) was used, which is designed for small-part imaging. Vessel 
compression was avoided, and oblique images of the vessel were captured using 
conductive gel, in accordance with recommendations from studies such as the one 
referenced16, due to the varying degrees of vascular impairment among patients. RA was 
always harvested from the non-dominant arm, and bilateral RA harvesting was not 
performed. As there was no control group, baseline assessments to evaluate normal 
vascular function are provided in the supplementary files, along with patient  
demographics. Although these patients already had vascular impairment, the graft arteries 
were chosen to be as healthy as possible to minimize complications after surgery and 
avoid shortening the graft's lifespan, thus preventing the need for another surgery within 
a short period. Data are available in the supplementary files. For the study, three groups 
were considered: mixed red and infrared LED (R + IR, n = 10), red LED (R, n = 10), and 
infrared LED (IR, n = 10).    

LED Irradiation in vitro on Radial Arteries   
After the arterial segments were collected for the study, they were transferred to   
HTK solution (commercially known as Custodiol® Contatti, manufactured by São 
Geraldo, Porto Alegre, Brazil), an intracellular crystalloid cardioplegia solution used in 
the surgical setting at: Clinical Hospital of Ribeirão Preto, University of São Paulo (USP), 
Brazil. This solution is used for myocardial protection in complex cardiac surgeries and 
organ preservation during transplant procedures. It aids in maintaining the integrity of 
vascular function after the removal of arterial segments, allowing for their safe transfer 
to the vascular biology laboratory.   
The arterial segments were positioned on a plate placed beneath an LED device. 
The distance between the LED panel and the bottom of the plate containing the arterial 
segments was approximately 5 mm. The total irradiation times were 94 seconds for the 
infrared LED blanket, 73 seconds for the mixed blanket (red + infrared), and 83 seconds  
for the red LED blanket.   
To ensure direct light exposure to all arterial segments, a pre-test was conducted 
using a light sensor (model E18-D80NK) to verify the effectiveness of the light delivery, 
given the 5 mm distance between the LED panel and the arterial segments. The LED 
blankets were procured from PlatinumLED Therapy Lights: BIOMAX 900 Series. Prior 
to use, all LEDs were tested in the Photobiophysics Laboratory at the Ribeirão Preto 
School of Philosophy, Sciences, and Letters, University of São Paulo, where the 
wavelengths, radiation angles, power, and power density of each LED were measured  
Vascular Function   
Vascular function was assessed using a wire myograph after the arterial segments 
were exposed to a preservation solution (costodiol) and subsequently immersed in a saline  
solution containing L-NAME (0.5 mg/ml). The L-NAME solution was used to induce 
vascular dysfunction for one minute, after which the segments were transferred to a 
modified Krebs-Henseleit solution at 4°C, with the following composition (in mM):  
NaCl, 130; KCl, 4.7; NaHCO₃, 14.9; KH₂PO₄, 1.18; MgSO₄, 1.17; glucose, 5.5; 
CaCl₂·2H₂O, 1.56; EDTA, 0.026.   
Radial artery rings (2 mm) from the R + IR, R, and IR groups were mounted in a 
myograph (model 620M; Danish Myo Technology – DMT, Copenhagen, Denmark)  

containing Krebs-Henseleit solution, gassed with 5% CO₂/95% O₂ to maintain a pH of  
7.4 for recording isometric tension. The rings were subjected to a basal passive force of 
5 mN, as defined by a voltage-response curve with rings subjected to tension between 0.5 
and 10 mN and stimulated with 10⁻⁷ M phenylephrine. A stabilization period of 45  
minutes was allowed.   
Following the stabilization period, potassium chloride (KCl, 120 mM) was used 
to test arterial contractility. Cumulative concentration-response curves were then 
constructed for phenylephrine (PE, 10⁻¹⁰ - 10⁻⁴ M), acetylcholine (ACh, 10⁻¹⁰ - 10⁻⁴ M), 
and sodium nitroprusside (SNP, 10⁻¹⁰ - 10⁻⁴ M).   
Lucigenin Chemiluminescence   
The superoxide anion (O₂•⁻), an important reactive oxygen species (ROS), 
generation in arteries from the R + IR, R, and IR groups was measured using a 
chemiluminescence assay with lucigenin as a nicotinamide electron acceptor and 
nicotinamide adenine dinucleotide phosphate (NADPH) as the substrate. Following  
stimulation, the arteries were washed and collected in a selective lysis buffer containing   
2 × 10⁻² M KH₂PO₄, 10⁻³ M EGTA, and protease inhibitors (1 μg/ml aprotinin, 1 μg/ml 
leupeptin, 1 μg/ml pepstatin, and 10⁻³ M phenylmethylsulfonyl fluoride [PMSF]). The 
samples were then transferred to glass tubes containing 990 μL of assay release solution   
[5 × 10⁻² M KH₂PO₄, 10⁻³ M EGTA, 1.5 × 10⁻³ M sucrose, pH 7.4] and 5 × 10⁻⁶ M 
lucigenin (98% purity, Sigma) for baseline measurement.   
NADPH (98% purity, 10⁻⁴ M; Sigma) was added to each sample, and the 
luminescence signal was measured for 30 cycles, each lasting 18 seconds, in a 
luminometer (Lumistar Galaxy, BMG Lab Technologies, Germany). A blank control was 
included, and baseline and blank readings were subtracted from the respective sample 
readings. The results are expressed as relative light units (RLU) per μg protein, as 
quantified by the BCA assay (Thermo Fisher, 23225).   
Nitrite Assay   
Nitrate/nitrite concentrations were measured using a Griess reagent kit (Thermo 
Fisher Scientific, catalog G7921) and nitrate reductase (Sigma-Aldrich, catalog N7265), 
following the manufacturer's instructions. Briefly, arterial segments were homogenized 
in 1x phosphate-buffered saline (PBS) and then centrifuged at 10,000 × g for 10 minutes 
at 4°C. Following centrifugation, 50 μL of the supernatant was transferred to a 96-well  

plate, and 50 μL of Griess reagent was added (composed of 5% H₃PO₄, 0.2% 
N(1Naphthyl) ethylenediamine, and 2% sulfanilamide). After a 10-minute incubation, the 
absorbance was measured at 540 nm using a FlexStation 3 microplate reader (Molecular 
Devices, San Jose, USA). Nitrite concentrations were determined from a standard curve 
prepared with nitrite solutions at final concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 
and 200 μmol/L.   
In Situ Measurement of Nitric Oxide (NO)   
Arteries were homogenized in 1x phosphate-buffered saline (PBS), and a 
5,6diaminofluorescein diacetate (DAF-2 DA, 5 μM, Sigma-Aldrich, catalog #50277) 
probe was added to the homogenate for a 30-minute incubation at 37°C. Nitric oxide (NO) 
levels were analyzed by fluorimetry using a FlexStation 3 microplate reader (Molecular 
Devices, San Jose, USA), with excitation and emission wavelengths set at 485 nm and 
538 nm, respectively. The results are expressed as relative light units (RLU) per milligram 
of protein.   
Immunoblotting   
The Intercellular Adhesion Molecule 1 (ICAM-1) expressionwas assessed by 
homogenizing samples in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% Nonidet 
P-40, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl 
sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml pepstatin A, 1 
μg/ml leupeptin, and 1 μg/ml aprotinin. The proteins were then collected. For 
immunoblotting, 30 μg of protein was separated by electrophoresis on 10% 
polyacrylamide gels and transferred to 0.22 μm nitrocellulose membranes. The 
membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline  
(TBS) containing 0.1% Tween 20 for 1 hour. Primary antibodies were incubated 
overnight at 4°C: anti-ICAM-1 (1:500 dilution; Santa Cruz, catalog sc-8439) and 
antiGAPDH (1:20,000 dilution; Sigma-Aldrich, catalog G9545).   
Statistical Analysis   
Continuous variables are presented as means ± standard deviation, and categorical 
variables are presented as frequencies and percentages. The normality of the variables  
was 
assessed 
using 
the 
Shapiro-Wilk 
test. 
Vascular 
changes 
following 
photobiomodulation were analyzed using Two-way ANOVA, followed by Tukey's post 

hoc test. A p-value < 0.05 (two-tailed) was considered statistically significant. All 
statistical analyses were performed using GraphPad Prism version 9.1.2 (GraphPad  
Software, San Diego, CA, USA).   
  
 

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