Biological Effects of Low Level Laser Therapy

Abstract

The use of low level laser to reduce pain, inflammation and edema, to promote wound, deeper tissues and nerves healing, and to prevent tissue damage has been known for almost forty years since the invention of lasers. This review will cover some of the proposed cellular mechanisms responsible for the effect of visible light on mammalian cells, including cytochrome c oxidase (with absorption peaks in the Near Infrared (NIR)). Mitochondria are thought to be a likely site for the initial effects of light, leading to increased ATP production, modulation of reactive oxygen species, and induction of transcription factors. These effects in turn lead to increased cell proliferation and migration (particularly by fibroblasts).

Introduction

Lasers (Light amplification by stimulated emission of radiation) are devices that typically generate electromagnetic radiation which are relatively uniform in wavelength, phase, and polarization, originally described by Theodore Maiman in 1960 in the form of a ruby laser ¹.

Laser is described as a source of light or radiation energy ². Low Level Laser (LLL) is a special type of laser that effects on biologic systems through non-thermal means ³. This area of investigation started with the work of Mester et al in 1967. They reported non-thermal effects of lasers on mouse hair growth ⁴.

According to Posten et al, properties of low level lasers are:

• a) Power output of lasers being 0.001- 0.1 Watts.

• b) Wave length in the range of 300-10,600 nm.

• c) Pulse rate from 0, meaning continuous to 5000 Hertz (cycles per second).

• d) Intensity of 0.01-10 W/cm² and dose of 0.01 to 100 J/ cm^{2 5}.

Most common methods of administration of LLL radiation include lasers such as ruby (694 nm), Ar (488 and 514 nm), He-Ne (632.8 nm), Krypton (521, 530, 568, and 647 nm), Ga-Al-As (805 or 650 nm), and Ga-As (904 nm) ³.

Low Level Laser therapy (LLLT) is the application of light to a biologic system to promote tissue regeneration, reduce inflammation and relieve pain. Unlike other medical laser procedures, LLLT does not have an ablative or thermal mechanism, but rather a photochemical effect which means the light is absorbed and cause a chemical change ⁶. The reason why the technique is termed low level is that the optimum levels of energy density delivered are low and it is not comparable to other forms of laser therapy as practiced for ablation, cutting, and thermal tissue coagulation ⁷.

The first law of photobiology explains that for a low power visible light to have any effect on a living biological system, the photons must be absorbed by electronic absorption bands belonging to some molecular photo-acceptors, which are called chromophores ⁸. The effective tissue penetration of light at 650 nm to 1200 nm is maximized. The absorption and scattering of light in tissue are both much higher in the blue region of the spectrum than the red, because the main tissue chromophores (hemoglobin and melanin) have high absorption bands at shorter wavelengths and tissue scattering of light is higher at shorter wavelengths. Water strongly absorbs infrared light at wavelengths greater than 1100 nm. Therefore, the use of LLLT in animals and patients almost exclusively utilizes red and near-infrared light (600-1100 nm) ⁹.

Mitochondrial Respiration and ATP

Current research about the mechanism of LLLT involves mitochondria ⁶. Cytochrome c oxidase (Cox) is a multicomponent membrane protein that contains a binuclear copper center (CuA) along with a heme binuclear center (a3-CuB), both of which facilitate the transfer of electrons from water soluble cytochrome c oxidase to oxygen. It is a terminal enzyme of the electron transport chain and plays a vital role in the bioenergetics of a cell ¹¹. It was proposed that Cox is the primary photoacceptor for the red-NIR range in mammalian cells because absorption spectra obtained for Cox in different oxidation states was found to be very similar to the action spectra for biological responses to light ¹⁰.

The absorption of photons by Cox leads to electronically excited states, and consequently can lead to quickening of electron transfer reactions ¹². More electron transport necessarily causes increased production of ATP ¹³.

The light induced increase in ATP synthesis and increased proton gradient lead to an increasing activity of the Na+/H+ and Ca2+/Na+ antiporters, and of all the ATP driven carriers for ions, such as Na+/K+ ATPase and Ca2+ pumps. ATP is the substrate for adenylcyclase, and therefore the ATP level controls the level of cAMP. Both Ca2+and cAMP are very important second messengers. Ca2+ regulates almost every process in the human body (muscle contraction, blood coagulation, signal transfer in nerves, gene expression, etc…) ⁷. Therefore the photoactivation of terminal enzymes, like Cox, plays a vital role in the activation of the diverse biological cascade observed subsequently to laser irradiation.

Nitric Oxide and LLLT

The activity of cytochrome c oxidase is inhibited by nitric oxide (NO) ^{14, 15}. This inhibition can be explained by a direct competition between NO and O2 for the reduced binuclear center CuB/a3 of cytochrome c oxidase, and is reversible ¹⁶. It was proposed that laser irradiation could reverse this inhibition by photodissociating NO from its binding sites ^{17, 18}. Because this coordinate binding is much weaker than a covalent bond, this dissociation is possible by LLL. The dissociation of NO from Cox increases the respiration rate ¹⁸. Light can indeed reverse the inhibition caused by NO binding to cytochrome oxidase, both in isolated mitochondria and in whole cells ¹⁹. LLL can also protect cells against NO-induced cell death ⁷.

Reactive Oxygen Species (ROS) and Gene Transcription

LLLT was reported to produce a shift in overall cell redox potential in the direction of greater oxidation ²⁰ and increased ROS generation and cell redox activity have been reported ^{21,22,23,24,25}. It has been proposed that the redox state of a cell regulates cellular signaling pathways that control gene expression. Modulation of the cellular redox state can activate or inhibit signaling pathways ¹¹. Several regulation pathways are mediated through the cellular redox state. Changes in redox state induce the activation of numerous intracellular signaling pathways, such as nucleic acid synthesis, protein synthesis, enzyme activation and cell cycle progression ²⁶.

These cytosolic responses may induce transcriptional changes. Several transcription factors have been recognized to regulate by changes in cellular redox state. Among them redox factor-1 (Ref-1)-dependent activator protein-1 (AP-1) (Fos and Jun), nuclear factor B (NF-B), p53, activating transcription factor/cAMP-response element-binding protein (ATF/ CREB), hypoxia-inducible factor (HIF)-1 and HIF-like factor are the most important factors ⁷.

Based on the ability of LLLT to modulate cellular metabolism and alter the transcription factors responsible for gene expression, it has been found to alter gene expression ²⁷.

LLL & Gene Expression

The gene expression profiles of human fibroblasts irradiated by low-intensity red light show that the irradiation can affect the expression of many genes that belong to different function categories ³⁶. Irradiation of LLL stimulates cell growth directly through regulation of the expression of genes related to cell proliferation and indirectly through regulation of the expression of genes related to cell migration and remodeling, DNA synthesis and repair, ion channel and membrane potential, and cell metabolism. Irradiation by red light also enhances cell proliferation by suppression of cell apoptosis ³⁶. Table 1 shows some of these genes.

Table 1. The effects of LLL on gene expression.

Name of genes	Role	Change	Mechanism
Mitogen-activated protein kinase 11 (MAPK11)	Proliferation	Up-regulation	Isoform of the p38 MAPK, which signaling pathway is involved in fibroblast growth factor2 induced proliferation28
Breakpoint cluster region (BCR)	Proliferation	upregulation	A GTPase-activating protein for Ras-related C3 botulinum toxin substrate 1 (RAC1) and Cell division control protein 42(CDC42) that promotes the exchange of RAC- or CDC42- bound GDP by GTP. Active RAC1 and CDC42 can suppress p21, leading to the upregulation of BCR gene, which can enhance cell growth 29
Platelet derived growth factor C (PDGF-C)	Proliferation	upregulation	A member of the PDGF/vascular endothelial growth factor family and its upregulation can induce mitogenic activity on several mesenchymal cell types 30
Serum response factor	Proliferation	upregulation	It contributes to mitogen-stimulated transcriptional induction of many immediate-early genes during the G0-G1 cell cycle transition and is also essential for cell cycle progression 31
Cullin 1	Prevent Proliferation	downregulation	The downregulated gene cullin 1 is an inhibitory regulator of the cell cycle. Cullin 1 is required for developmentally programmed transitions from the G1 phase to the G0 phase of the cell cycle or the apoptotic pathway, the mutation of which leads to the acceleration of G1 to S phase progression 32
Heat shock 70kD protein 1A Caspase 6 Stress induced-phosphoprotein 1	Apoptosis	downregulation	Apoptpsis
NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2	Energy metabolism and respiratory chain	upregulation	It is one of the peptides of mitochondria respiratory complex I that transfer electrons from NADH to the respiratory chain 33.
ATP synthase, H þ transporting, mitochondrial F0 complex, subunitd	Energy metabolism and respiratory chain	upregulation	ATP5H belongs to the respiratory complex V (F1F0 -ATPase assembly), which catalyzes ATP synthesis 34
Electron-transfer-flavoprotein, beta polypeptide	Energy metabolism and respiratory chain	upregulation	Electron-transfer-flavoprotein b polypeptide (ETFB) is a subunit of ETF that serves as a specific electron acceptor for several dehydrogenases including acyl-CoA dehydrogenases that function in fatty acid b oxidation 35

The Usage of LLLT

The temporomandibular disorders (TMDs) have been identified as the most important cause of pain in the facial region. Low Level laser therapy (LLLT) has demonstrated to have analgesic, anti-inflammatory and biostimulating effects. The LLLT is a noninvasive, quick and safe, non-pharmaceutical intervention that may be beneficial for patients with TMDs ³⁷.

Another study proposed a novel combination of neural regeneration techniques for the repair of damaged peripheral nerves. A biodegradable nerve conduit containing genipin-cross-linked gelatin was annexed using beta-tricalcium phosphate (TCP) ceramic particles (genipin-gelatin-TCP, GGT) to bridge the transection of a 15mm sciatic nerve in rats. Electrophysiological measurements (peak amplitude and area) illustrated by compound muscle action potential (CMAP) curves demonstrated that laser stimulation significantly improved nerve function and reduced muscular atrophy. Histomorphometric assessments revealed that laser stimulation accelerated nerve regeneration over a larger area of neural tissue, resulting in axons of greater diameter and myelin sheaths of greater thickness than that observed in rats treated with nerve conduits alone ³⁸.

Summary

The molecular and cellular mechanisms of LLLT suggest that photons are absorbed by the mitochondria. They stimulate more ATP production and low levels of ROS, which then activates transcription factors, such as NF-κB, to induce many gene transcript products responsible for the beneficial effects of LLLT. ROS are well known to stimulate cellular proliferation of low levels, but inhibit proliferation and kill cells at high levels. Nitric oxide is also involved in LLLT, and may be photo-released from its binding sites in the respiratory chain and elsewhere. It is possible that NO release in low amounts by low dose light may be beneficial.

Further advances in the mechanistic understanding of LLLT will continue to be made in the near future. These advances will lead to greater acceptance of LLLT in main- stream medicine and may lead to LLLT being used for serious diseases such as stroke, heart attack and degenerative brain diseases.

Please cite this article as follows:

Farivar S, Malekshahabi T, Shiari R. Biological Effects of Low Level Laser Therapy. J Lasers Med Sci 2014;5(2):58-62