Inflammation

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About Author manuscripts Submit a manuscript HHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
AIMS Biophys. Author manuscript; available in PMC 2017 Jul 24.
Published in final edited form as:
AIMS Biophys. 2017; 4(3): 337–361.

Published online 2017 May 19. doi:  10.3934/biophy.2017.3.337

PMCID: PMC5523874
NIHMSID: NIHMS879874

Mechanisms and applications of the anti-inflammatory effects of photobiomodulation

Michael R Hamblin1,2,3,*
1Wellman Center for Photomedicine, Massachusetts General Hospital, BAR414, 40 Blossom Street, Boston, MA 02114, USA
2Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA
3Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA

Abstract

Photobiomodulation (PBM) also known as low-level level laser therapy is the use of red and near-infrared light to stimulate healing, relieve pain, and reduce inflammation. The primary chromophores have been identified as cytochrome c oxidase in mitochondria, and calcium ion channels (possibly mediated by light absorption by opsins). Secondary effects of photon absorption include increases in ATP, a brief burst of reactive oxygen species, an increase in nitric oxide, and modulation of calcium levels. Tertiary effects include activation of a wide range of transcription factors leading to improved cell survival, increased proliferation and migration, and new protein synthesis. There is a pronounced biphasic dose response whereby low levels of light have stimulating effects, while high levels of light have inhibitory effects. It has been found that PBM can produce ROS in normal cells, but when used in oxidatively stressed cells or in animal models of disease, ROS levels are lowered. PBM is able to up-regulate anti-oxidant defenses and reduce oxidative stress. It was shown that PBM can activate NF-kB in normal quiescent cells, however in activated inflammatory cells, inflammatory markers were decreased. One of the most reproducible effects of PBM is an overall reduction in inflammation, which is particularly important for disorders of the joints, traumatic injuries, lung disorders, and in the brain. PBM has been shown to reduce markers of M1 phenotype in activated macrophages. Many reports have shown reductions in reactive nitrogen species and prostaglandins in various animal models. PBM can reduce inflammation in the brain, abdominal fat, wounds, lungs, spinal cord.

1. Introduction

Photobiomodulation (PBM) was discovered almost 50 years ago by Endre Mester in Hungary []. For most of this time PBM was known as “low-level laser therapy” as ruby laser (694 nm) and HeNe lasers (633 nm) were the first devices used. Recently a consensus decision [] was taken to use the terminology “PBM” since the term “low-level” was very subjective, and it is now known that actual lasers are not required, as non-coherent light-emitting diodes (LEDs) work equally well []. For much of this time the mechanism of action of PBM was unclear, but in recent years much progress has been made in elucidating chromophores and signaling pathways [].

Most of the early work in this field was carried out with various kinds of lasers, and it was thought that laser light had some special characteristics not possessed by light from other light sources such as sunlight, fluorescent or incandescent lamps and now LEDs. However all the studies that have been done comparing lasers to equivalent light sources with similar wavelength and power density of their emission, have found essentially no difference between them.

Many wavelengths in the red (600–700 nm) and near-infrared (NIR, 770–1200 nm) spectral regions have shown positive results, however there is a region in between (700–770 nm) where broadly speaking, the results are likely to be disappointing. Recently blue and green wavelengths have also begun to be explored [] but they have major problems with penetration depth. It is accepted that penetration of light into tissue is governed by both absorption and scattering by molecules and structures present in tissue. Both absorption and scattering become significantly less as the wavelength gets longer, so the penetration depth of NIR is maximal about 810 nm, and at longer wavelengths water becomes an important absorber and penetration depth gets shorter again [].

The “biphasic dose response” describes a situation in which there is an optimum value of the “dose” of PBM most often defined by the energy density (J/cm2) [,]. It has been consistently found that when the dose of PBM is increased a maximum response is reached at some value, and if the dose in increased beyond that maximal value, the response diminishes, disappears and it is even possible that negative or inhibitory effects are produced at very high fluences.

2. Chromophores Responsible for Photobiomodulation

2.1. Cytochrome c oxidase in mitochondria

Cytochrome c oxidase (CCO) is unit IV in the mitochondrial electron transport chain. It transfers one electron (from each of four cytochrome c molecules), to a single oxygen molecule, producing two molecules of water. At the same time the four protons required, are translocated across the mitochondrial membrane, producing a proton gradient that the ATP synthase enzyme needs to synthesize ATP. CCO has two heme centers (a and a3) and two copper centers (CuA and CuB). Each of these metal centers can exist in an oxidized or a reduced state, and these have different absorption spectra, meaning CCO can absorb light well into the NIR region (up to 950 nm) []. Tiina Karu from Russia was the first to suggest [,], that the action spectrum of PBM effects matched the absorption spectrum of CCO, and this observation was confirmed by Wong-Riley et al in Wisconsin []. The assumption that CCO is a main target of PBM also explains the wide use of red/NIR wavelengths as these longer wavelengths have much better tissue penetration than say blue or green light which are better absorbed by hemoglobin. The most popular theory to explain exactly why photon absorption by CCO could led to increase of the enzyme activity, increased oxygen consumption, and increased ATP production is based on photodissociation of inhibitory nitric oxide (NO) []. Since NO is non-covalently bound to the heme and Cu centers and competitively blocks oxygen at a ratio of 1:10, a relatively low energy photon can kick out the NO and allow a lot of respiration to take place [].

2.2. Light gated ion channels and opsins

More recently it has become apparent that another class of photoreceptors, must be involved in transducing cellular signals, particularly responding to blue and green light. Thee photoreceptors have been proposed to be members of the family of light-sensitive G-protein coupled receptors known as opsins (OPN). Opsins function by photoisomerization of a cis-retinal co-factor leading to a conformational change in the protein. The most well known opsin is rhodopsin (OPN1), which is responsible for mediating vision in the rod and cone photoreceptor cells in the mammalian retina. There are other members of the opsin family (OPN2-5), which are expressed in many other tissues of the body including the brain []. One of the best-defined signaling events that occurs after light-activation of opsins, is the opening of light-gated ion channels such as members of the transient receptor potential (TRP) family of calcium channels []. TRP channels are now known to be pleiotropic cellular sensors mediating the response to a wide range of external stimuli (heat, cold, pressure, taste, smell), and involved in many different cellular processes []. Activation of TRP causes non-selective permeabilization (mainly of the plasma membrane) to calcium, sodium and magnesium []. It is now known that TRP channel proteins are conserved throughout evolution and are found in most organisms, tissues, and cell-types. The TRP channel superfamily is now classified into seven related subfamilies: TRPC, TRPM, TRPV, TRPA, TRPP, TRPML, and TRPN []. Light-sensitive ion channels are based on an opsin chromophore (isomerization of a cis-retinal molecule to the trans configuration) as illustrated in Drusophila photoreceptors [].

We have shown that blue or green light (but not red or 810 nm NIR) increased intracellular calcium in adipose derived stem cells, that could be blocked by ion channel inhibitors [].

2.3. Flavins and flavoproteins

There is another well-known family of biological chromophores called cryptochromes. These proteins have some sequence similarity to photolyases [], which are blue light responsive enzymes that repair DNA damage in bacteria caused by UV exposure []. Cryptochromes rely on a flavin (flavin adenine dinucleotide, FAD) or a pterin (5,10-methenyltetrahydrofolic acid) to actually absorb the light (again usually blue or green). Cryptochromes have been studied mainly in plants and insects. Recent evidence has emerged that mammalian cryptochromes are important in regulation of the circadian clock. It is thought that human cryptochromes (CRY1 and CRY2) send signals via part of the optic nerve to the suprachiasmatic nucleus (SCN) in the brain, which is the master regulator of the CLOCK system to entrain biological responses to the light-dark cycle []. However the situation is complicated because retinal ganglion cells containing melanopsin (OPN4) are also involved in photoentrainment []. Studies are still ongoing to investigate this redundancy [].

It should be emphasized that compared to CCO and mitochondria, evidence is still emerging concerning the extent to which opsins, cryptochomes and light-gated ion channels (which may be widely expressed in many different cell types) could be responsible for PBM effects. If their role is significant it is likely to be in the blue and green spectral regions. Further research will be necessary to explore their role in anti-inflammatory effects, wound healing and tissue regeneration.

2.4. Water as a chromophore and heat-gated ion channels

Since the biological effects of light continue to be observed, as the wavelength increases in the infra-red region (>1000 nm), beyond those known to be absorbed by CCO, it is now thought likely that an alternative chromophore must be responsible. The obvious candidate for this alternative chromophore is water molecules whose absorption spectrum has peaks at 980 nm, and also at most wavelengths longer than 1200 nm. Moreover, water is by the far the most prevalent molecule in biological tissue (particularly considering its low molecule weight = 18). At present the proposed mechanism involves selective absorption of IR photons by structured water layers (also known as interfacial water) [] or water clusters [], at power levels that are insufficient to cause any detectable bulk-heating of the tissue. A small increase in vibrational energy by a water cluster formed in or on a sensitive protein such as a heat-gated ion channel, could be sufficient to perturb the tertiary protein structure thus opening the channel and allowing modulation of intracellular calcium levels []. Pollack has shown that interfacial water can undergo charge separation when it absorbs visible or NIR light []. This charge separation (equivalent to localized pH changes) could affect the conformation of proteins []. It has also been suggested that PBM could reduce the viscosity of interfacial water within the mitochondria, and allow the F0F1 ATP synthase, which rotates as a nanomotor to turn faster []. It should be noted here that the first regulatory approvals of PBM were gained as a 510 K device “equivalent to an non-heating IR lamp” []. While the involvement of water as a chromophore may still be considered hypothetical it is difficult to think of another explanation for the beneficial of PBM at wavelengths between 1000 nm all the way to 10,000 nm (carbon dioxide laser).

The molecular chromophores discussed above are graphically summarized in Figure 1.

Figure 1

Chromophores in PBM. Cytochrome c oxidase in respiratory chain absorbs mainly red (and NIR) light by heme and copper; Heat-gated TRP ion channels absorb NIR (and blue light) via structured water; opsins absorb mainly blue/green light via cis-retinal; 

3. Effects of PBM on Reactive Oxygen Species and Oxidative Stress

3.1. PBM increases ROS in normal cells

When PBM stimulates CCO activity in normal healthy cells, the resulting increase in mitochondrial membrane potential (MMP) above normal baseline levels, leads to a brief and rather modest increase in generation of reactive oxygen species (ROS) []. However this brief burst of ROS caused by 3 J/cm2 of 810 nm laser (Figure 2A) was shown to be sufficient to activate the redox-sensitive transcription factor, NF-kB in embryonic fibroblasts [] (Figure 2B). Addition of the anti-oxidant N-acetyl-cysteine to the cells could block the NK-kB activation (Figure 2C), but not the increase in cellular ATP caused by the mitochondrial stimulation (Figure 2D). In primary cultured cortical neurons [], 810 nm laser produced a biphasic dose response in ATP production (Figure 3A) and MMP (Figure 3B) with a maximum at 3 J/cm2. At a high dose (30 J/cm2) the MMP was actually lowered below baseline. Interestingly the dose-response curve between fluence (J/cm2) and ROS production showed two different maxima (Figure 3C). One of these maxima occurred at 3 J/cm2 where the MMP showed its maximum increase. The second maximum in ROS production occurred at 30 J/cm2where the MMP had been reduced below baseline. At a value between these two fluences (10 J/cm2) a dose at which the MMP was approximately back to baseline, there was not much ROS generation. These data are very good examples of the “biphasic dose response” or “Arndt-Schulz curve” which is often discussed in the PBM literature [,].

Figure 2

NFkB is activated by PBM induced ROS in embryonic fibroblasts. (A) Intracellular ROS measured by DCDHF fluorescence; (B) NF-kB activation measured by a luciferase assay; (C) NF-kB activation is inhibited by antioxidants; (D) ATP increase is not affected 

Figure 3

Dose response of 810 nm laser in cortical neurons. (A) ATP production as a function of fluence; (B) Mitochondrial membrane potential (JC1 red/green ratio); (C) Mitochondrial ROS. Figure adapted from data in [].

Thus it appears that ROS can be generated within mitochondria when the MMP is increased above normal values and also when it is decreased below normal values. It remains to be seen whether these two kinds of PBM-generated ROS are identical or not. One intriguing possibility is that whether the ROS generated by PBM is beneficial or detrimental may depend on the rate at which it is generated. If superoxide is generated in mitochondria at a rate that allows superoxide dismutase (SOD) to detoxify it to hydrogen peroxide, then the uncharged H2O2 can diffuse out of the mitochondria to activate beneficial signaling pathways, while if superoxide is generated at a rate or at levels beyond the ability of SOD to deal with it, then the charged superoxide may build up inside mitochondria and damage them.

3.2. PBM reduces ROS in oxidative stressed cells and tissues

Notwithstanding, the ability of PBM to produce a burst of ROS in normal cells, it is well-accepted that PBM when as a treatment for tissue injury or muscle damage is able to reduce markers of oxidative stress [,,]. How can these apparently contradictory findings be reconciled? A study attempted to answer this question []. Primary cultured cortical neurons were treated with one of three different interventions, all of which were chosen from literature methods of artificially inducing oxidative stress in cell culture. The first was cobalt chloride (CoCl2), which is used as a mimetic for hypoxia and works by a Fenton reaction producing hydroxyl radicals []. The second was direct treatment with hydrogen peroxide. The third was treatment with the mitochondrial complex I inhibitor, rotenone []. All three of these different treatments increased the intracellular mitochondrial ROS as judged by Cell-Rox Red (Figure 4A), and at the same time lowered the MMP as measured by tetramethyl-rhodamine methyl ester (TMRM) (Figure 4B). PBM (3 J/cm2 of 810 nm laser) raised the MMP back towards baseline, while simultaneously reducing the generation of ROS in oxidatively stressed cells (while slightly increasing ROS in normal cells). In control cells (no oxidative stress), PBM increased MMP above baseline and still produced a modest increase in ROS.

Figure 4

PBM reduces oxidative stress in cortical neurons. Oxidative stress was induced by three different treatments (cobalt chloride, hydrogen peroxide, rotenone) and cells were treated with 3 J/cm2 810 nm. (A) Mitochondrial ROS, (B) Mitochondrial membrane potential 

Since most laboratory studies of PBM as a therapy have looked at various animal models of disease or injury, it is not surprising that most workers have measured reduction in tissue markers of oxidative stress (TBARS) after PBM [,]. There have been a lot of studies looking at muscles. In humans, especially in athletes, high-level exercise produces effects in muscles characterized by delayed-onset muscle soreness, markers of muscle damage (creatine kinase), inflammation and oxidative stress.

One cellular study by Macedo et al [] used muscle cells isolated from muscular dystrophy mice (mdx LA 24) and found that 5 J/cm2 of 830 nm increased the expression levels of myosin heavy chain, and intracellular [Ca2+]i. PBM decreased H2O2production and 4-HNE levels and also GSH levels and GR and SOD activities. The mdx cells showed significant increase in the TNF-? and NF?B levels, which were reduced by PBM.

While it is highly likely that the effects of PBM in modulating ROS are involved in the anti-inflammatory effects of PBM, it would be dangerous to conclude that that is the only explanation. Other signaling pathways (nitric oxide, cyclic AMP, calcium) are also likely to be involved in reduction of inflammation.

4. Effects of PBM on NF-kB

4.1. PBM activates NF-kB in normal cells

As mentioned above we found [] that PBM (3 J/cm2 of 810 nm laser) activated NF-kB in embryonic fibroblasts isolated from mice that had been genetically engineered to express firefly luciferase under control of an NF-kB promoter. Although it is well-known that NF-kB functions as a pro-inflammatory transcription factor, but on the other hand it is also well known that in clinical practice or in laboratory animal studies) PBM has a profound anti-inflammatory effect in vivo. This gives rise to another apparent contradiction that must be satisfactorily resolved.

4.2. PBM reduces levels of pro-inflammatory cytokines in activated inflammatory cells

Part of the answer to the apparent contradiction highlighted above, was addressed in a subsequent paper []. We isolated primary bone marrow-derived dendritic cells (DCs) from the mouse femur and cultured them with GM-CSF. When these cells were activated with the classical toll-like receptor (TLR) agonists, LPS (TLR4) and CpG oligodeoxynucleotide (TLR9), they showed upregulation of cell-surface markers of activation and maturation such as MHC class II, CD86 and CD11c as measured by flow cytometry. Moreover IL12 was secreted by CpG-stimulated DCs. PBM (0.3 or 3 J/cm2of 810 nm laser) reduced all the markers of activation and also the IL12 secretion. Figure 5.

Figure 5

PBM reduces inflammatory markers in activated murine DCs in vitro. (A) Flow cytometry was used to measure MHC class II, CD86, CD11c (dexamethasone was used as positive control); (B) Secreted IL12 measured by ELISA. Figure adapted from data in [].

Yamaura et al [] tested PBM (810 nm, 5 or 25 J/cm2) on synoviocytes isolated from rheumatoid arthritis patients. They applied PBM before or after addition of tumor necrosis factor-? (TNF-?). mRNA and protein levels of TNF-? and interleukins (IL)-1beta, and IL-8 were reduced (especially by 25 J/cm2).

Hwang et al [] incubated human annulus fibrosus cells with conditioned medium obtained from macrophages (THP-1 cells) containing proinflammatory cytokines IL1?, IL6, IL8 and TNF-?. They compared 405, 532 and 650 nm at doses up to 1.6 J/cm2. They found that all wavelengths reduced IL8 expression and 405 nm also reduced IL6.

The “Super-Lizer” is a Japanese device that emits linear polarized infrared light. Imaoka et al [] tested it against a rat model of rheumatoid arthritis involving immunizing the rats with bovine type II collagen, after which they develop autoimmune inflammation in multiple joints. The found reductions in IL20 expression in histological sections taken from the PBM-treated joints and also in human rheumatoid fibroblast-like synoviocyte (MH7A) stimulated with IL1?.

Lim et al [] studied human gingival fibroblasts (HGF) treated with lipopolysaccharides (LPS) isolated from Porphyromonas gingivalis. They used PBM mediated by a 635 nm LED and irradiated the cells + LPS directly or indirectly (transferring medium from PBM treated cells to other cells with LPS). Both direct and indirect protocols showed reductions in inflammatory markers (cyclooxygenase-2 (COX2), prostaglandin E2 (PGE2), granulocyte colony-stimulating factor (GCSF), regulated on activated normal T-cell expressed and secreted (RANTES), and CXCL11). In the indirect irradiation group, phosphorylation of C-Raf and Erk1/2 increased. In another study [] the same group used a similar system (direct PBM on HGF + LPS) and showed that 635 nm PBM reduced IL6, IL8, p38 phosphorylation, and increased JNK phosphorylation. They explained the activation of JNK by the growth promoting effects of PBM. Sakurai et al reported [] similar findings using HGF treated with Campylobacter rectus LPS and PBM (830 nm up to 6.3 J/cm2) to reduce levels of COX2 and PGE2. In another study [] the same group showed a reduction in IL1? in the same system.

4.3. Effects of PBM on macrophage phenotype

Another very interesting property of PBM is its ability to change the phenotype of activated cells of the monocyte or macrophage lineage. These cells can display two very different phenotypes depending on which pathological situation the cells are faced with. The M1 phenotype (classically activated) applies to macrophages that are faced with a situation in which bacteria or other pathogens need to be killed, or alternatively tumor cells need to be destroyed. Inducible nitric oxide synthase is a hallmark of the M1 phenotype and nitric oxide secretion is often measured. On the other hand the M2 phenotype (alternatively activated) applies to macrophages that are involved in disposal of cellular or protein debris and stimulation of healing by angiogenesis. The M2 phenotype produces arginase, an enzyme that inhibits NO production and allows them to produce ornithine, a precursor of hydroxyproline and polyamines []. The markers of these two phenotypes of activated macrophage have some aspects in common, but also show many aspects that are very different []. It should be noted that this concept of M1 and M2 activation states, applies to other specialized macrophage type cells that are resident in different tissues, such as microglia in brain [], alveolar macrophages in lung [], Kuppfer cells in liver [], etc.

Fernandes et al used J774 macrophage-like cells activated with interferon-? and LPS to produce a MI phenotype and compared 660 nm and 780 nm laser. They found that both wavelengths reduced TNF-?, COX-2 and iNOS expression, with the 780 nm being somewhat better []. Silva et al used RAW264.7 macrophages to test two wavelengths (660 nm and 808 nm) at a range of fluences (11-214 J/cm2) []. They found increases in NO release with 660 nm at the higher fluences. von Leden et al carried out an interesting study looking at the effects of PBM on microglia and their interaction with cortical neurons []. They used both primary microglia isolated from mouse brains and the BV2 mouse microglial cell line and compared four fluences (0.2, 4, 10, and 30 J/cm2, at 808 nm. Fluences between 4 and 30 J/cm2 induced expression of M1 markers in microglia. Markers of the M2 phenotype, including CD206 and TIMP1, were observed at lower energy densities of 0.2–10 J/cm2. In addition, co-culture of PBM or control-treated microglia with primary neuronal cultures demonstrated a dose-dependent effect of PBM on microglial-induced neuronal growth and neurite extension. This suggests that the benefits of PBM on neuroinflammation may be more pronounced at lower overall doses. The same group went on to show that M1 activated macrophages receiving PBM (660 nm laser) showed significant decreases in CCL3, CXCL2 and TNF? mRNA expression 4 h after irradiation []. However, 24 h after irradiation, M1 macrophages showed increased expression of CXCL2 and TNF? genes. M1 activated macrophages irradiated with 780 nm showed a significant decrease in CCL3 gene expression 4h after irradiation. These data could explain the anti-inflammatory effects of LLLT in wound repair.

5. Effects of PBM on Inflammation in Animal Models of Disease

This section will cover some of the most important medical indications where PBM has been shown in laboratory studies to be effective (at least partly) by its pronounced anti-inflammatory effects. Figure 6 shows a graphical summary of the anti-inflammatory applications of PBM in experimental animal models.

Figure 6

Animal models in which the anti-inflammatory effects of PBM have been shown. Acute traumatic brain injury; experimental autoimmune encephalomyelitis; spinal cord injury; wound healing; muscle exercise and recovery; inflammatory pain in paw; abdominal 

5.1. Wound healing

Many papers have demonstrated the efficacy of PBM in stimulating wound healing. In animal models these studies have generally been on acute wounds [], while in clinical trials they are often been concerned with chronic non-healing wounds such as diabetic ulcers []. Gupta et al [] tested PBM using a superpulsed 904 nm laser on burn wounds in rats. They found faster healing, reduced inflammation (histology), decreased expression of TNF-? and NF-kB, and up-regulated expression of VEGF, FGFR-1, HSP-60, HSP-90, HIF-1? and matrix metalloproteinases-2 and 9 compared to controls. It is intriguing to speculate that the effects of PBM on wound healing (especially the use of for chronic non-healing wounds) could involve both pro-inflammatory effects and anti-inflammatory effects. This seemingly contradictory statement may be possible due to the recent discovery of resolvins and protectins, which are multifunctional lipid mediators derived from omega-3 polyunsaturated fatty acids []. If resolvins were produced as a result of the brief acute inflammation induced by application of PBM to chronic wounds, then it has been already shown that resolvins can hasten the healing of diabetic wounds in mice []. Resolvins have been shown to reduce tumor necrosis factor-?, interleukin-1?, and neutrophil platelet-endothelial cell adhesion molecule-1 in a mouse burn wound model [].

5.2. Arthritis

In humans, arthritis is most often caused by a degenerative process occurring in osteoarthritis, or an autoimmune process occurring in rheumatoid arthritis. Both are characterized by pronounced inflammatory changes in the joint and even systemically. Different animal models are produced to mimic these diseases, but a common approach is to inject the sterile preparation of yeast cell walls known as zymosan into the knee joints of rats.

Castano et al [] used this zymosan-induced arthritis model to study the effects of two different fluences of 810 nm laser (3 and 30 J/cm2) delivered at two different power densities (5 and 50 mW/cm2). PBM was delivered once a day for 5 days commencing after zymosan injection, and the swelling in the knee was measured daily. Prostagladin E2 (PGE2) was measured in the serum. They found that 3 out of the 4 sets of parameters were approximately equally effective in reducing swelling and PGE2, but the ineffective set of parameters was 3 J/cm2 delivered at 50 mW/cm2 which only took 1 min of illumination time. The conclusion was, that the illumination time was important in PBM, and if that time was too short, then the treatment could be ineffective.

Moriyama et al [] used a transgenic mouse strain (FVB/N-Tg(iNOS-luc) that had been engineered to express luciferase under control of the inducible nitric oxide synthase promoter, to allow bioluminescence imaging of PBM of the zymosal-induced arthritis model in mice knees. They compared the same fluence of 635, 660, 690, and 905 nm (CW0 and 905 nm (short pulse). Animals younger than 15 weeks showed mostly reduction of iNOS expression, while older animals showed increased iNOS expression. Pulsed 905 nm also increased iNOS expression.

Pallotta et al [] used a model where carageenan was injected into the rat knee and tested 810 nm laser at 1, 3, 6 or 10 J/cm2. Rats were sacrificed after 6 or 12 hours and the joint tissue removed. PBM was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Gene expression of both COX-1 and 2 were significantly enhanced by laser irradiation while PGE2 production was inhibited. These apparently contradictory results require more study to fully explain.

5.3. Muscles

One of the most robust applications of PBM, is its effects on muscles [,]. PBM can potentiate muscular performance especially when applied to the muscles 3 hours before exercise []. PBM can also make exercise-training regimens more effective. It is not therefore surprising that PBM can also help to heal muscle injuries, not to mention reducing muscle pain and soreness after excessive exercise. Many of the animal studies that have been done have looked at markers of inflammation and oxidative stress in muscle tissue removed from sacrificed animals. For instance, Silveira et al [] caused a traumatic muscle injury by a single blunt-impact to the rat gastrocnemius muscle. PBM (850 nm, 3 or 5 J/cm2) was initiated 2, 12, and 24? h after muscle trauma, and repeated for five days. The locomotion and muscle function was improved by PBM. TBARS, protein carbonyls, superoxide dismutase, glutathione peroxidase, and catalase, were increased after muscle injury, these increases were prevented by PBM. PBM prevented increases in IL-6 and IL-10 and reversed the trauma-induced reduction in BDNF and VEGF.

5.4. Inflammatory pain

There have been many studies that have looked at the effects of PBM on pain in animal models. Some studies have looked at sensitivity to pain [] using the von Frey filaments (a graded set of fibers of increasing stiffness and when the animal feels the pressure it withdraws its foot []).

Some studies have looked at animal models of neuropathic pain such as the “spared nerve injury” []. This involves ligating two out of three branches of the sciatic nerve in rats and causes long lasting (>6 months) mechanical allodynia []. Kobelia Ketz et al found improvements in pain scores with PBM (980? nm applied to affected hind paw 1 W, 20 s, 41 cm above skin, power density 43.25 ?mW/cm2, dose 20 J). They also found lower expression of the proinflammatory marker (Iba1) in microglia in the dorsal root ganglion, gracile nucleus, dorsal column and dorsal horn. The M1/M2 balance of the macrophage phenotype was switched from M1 to M2 by PBM, as judged by relative staining with anti-CD86 (M1) and anti-CD206 (M2).

Martins et al looked at the effect of PBM on a model of inflammatory pain []. This involved injecting complete Freund’s adjuvant (CFA) into the mouse paw, and produces hyperalgesia and elevated cytokine levels (TNF-?, IL-1?, IL-10). They found that LEDT (950-nm, 80 mW/cm2, 1, 2 or 4 J/cm2) applied to the plantar aspect of the right hind limb, reduced pain, increased the levels of IL-10 prevented TBARS increase in both acute and chronic phases, reduced protein carbonyl levels and increased SOD and CAT activity in the acute phase only.

5.5. Lung inflammation

Aimbire and his laboratory in Brazil have carried out several studies on the use of PBM to reduce acute lung inflammation (ALI) in various animal models. In a mouse model of lung inflammation caused either by inhalation of lipolysaccharide or intranasal administration of TNF? they analyzed the bronchoalveolar lavage fluid (BALF). PBM (660 nm, 4.5 J/cm2) was administered to the skin over the right upper bronchus 15 min after ALI induction. PBM attenuated the neutrophil influx and lowered TNF? in BALF. In alveolar macrophages, PBM increased cAMP and reduced TNF? mRNA.

They also studied a different model of ALI caused by intestinal ischemia and reperfusion (I/R), that produces an analogue of acute respiratory distress syndrome (ARDS) []. Rats were subjected to superior mesenteric artery occlusion (45 min) and received PBM (660 nm, 7.5 J/cm2) carried out by irradiating the rats on the skin over the right upper bronchus for 15 and 30 min, and rats were euthanized 30 min, 2, or 4 h later. PBM reduced lung edema, myeloperoxisdase activity, TNF-? and iNOS, LLLT increased IL-10 in the lungs of animals subjected to I/R.

A third animal model was related to asthma []. Mice were sensitized to ovalbumin (OVA), and then challenged by a single 15-min exposure to aerosolized OVA. PBM was applied as above (660 nm, 30 mW, 5.4 J). Bronchial hyper-responsiveness (as measured by dose response curves to acetylcholine) was reduced by PBM as well as reductions in eosinophils and eotaxin. PBM also diminished expression of intracellular adhesion molecule and Th2 cytokines, as well as signal transducer and activator of transduction 6 (STAT6) levels in lungs from challenged mice. Recently Rigonato-Oliveira et al. presented a study that concluded that the reduced lung inflammation and the positive effects of PBM on the airways appear to be mediated by increased secretion of the anti-inflammatory cytokine IL-10, and reduction of mucus in the airway [].

5.6. Traumatic brain injury

In recent years the use of PBM as a treatment for traumatic brain injury [,], and other brain disorders including stroke, neurodegenerative diseases and even psychiatric disorders has increased markedly []. It is thought that the actions of NIR light shone on the head and penetrating into the brain are multi-factorial, but one clear effect is the anti-inflammatory action of transcranial PBM. This was shown by a series of mouse experiments conducted by Khuman et al []. They used the controlled cortical impact model of TBI and delivered PBM (800? nm) was applied directly to the contused parenchyma or transcranially in mice beginning 60–80?min after CCI. Injured mice treated with 60?J/cm2 (500 ?mW/cm2 × 2? min) had improved latency to the hidden platform and probe trial performance in the Morris water maze. PBM in open craniotomy mice reduced the number of activated microglia in the brain at 48? h (21.8 ± 2.3 versus 39.2 ± 4.2 IbA-1 + cells/field).

5.7. Spinal cord injury

Spinal cord injury (SCI) is another promising area of central nervous system injury that could be benefited by PBM. Veronez et al [] used a rat model of SCI involving a contusion produced by a mechanical impactor (between the ninth and tenth thoracic vertebrae), with a pressure of 150 kdyn. Three different doses of PBM (808-nm laser) were tested: 500 J/cm2, 750 J/cm2 and 1000 J/cm2 delivered daily for seven days. Functional preformance and tactile sensitivity were improved after PBM, at 1000 J/cm2. PBM at 750 and 1000 J/cm2 reduced the lesion volume and also reduced markers of inflammation (lower CD-68 protein expression).

5.8. Autoimmune diseases

Experimental autoimmune encephalomyelitis (EAE) is the most commonly studied animal model of multiple sclerosis (MS), a chronic autoimmune demyelinating disorder of the central nervous system. Immunomodulatory and immunosuppressive therapies currently approved for the treatment of MS slow disease progression, but do not prevent it. Lyons et al [] studied a mouse model of EAE involving immunization with myelin oligodendrocyte glycoprotein (MOG35-55). They treated the female C57BL/6 mice with PBM (670 nm) for several days in different regimens. In addition to improved muscular function, they found down-regulation of inducible nitric oxide synthase (iNOS) gene expression in the spinal cords of mice as well as an up-regulation of the Bcl-2 anti-apoptosis gene, an increased Bcl-2:Bax ratio, and reduced apoptosis within the spinal cord of animals over the course of disease. 670 nm light therapy failed to ameliorate MOG-induced EAE in mice deficient in iNOS, confirming a role for remediation of nitrosative stress in the amelioration of MOG-induced EAE by 670 nm mediated photobiomodulation.

5.9. Abdominal fat

Yoshimura et al [] looked at a mouse model of obesity and type 2 diabetes []. Four weeks old male adult C57BL/6 mice were fed a hypercaloric high-fat diet (40% calories derived from fat) for eight weeks to induce obesity and hyperglycemia. Over a period of four weeks mice were exposed to six irradiation sessions using an 843 nm LED (5.7 J cm?2, 19 mW cm?2). Non-irradiated control mice had areas of inflammation in their abdominal fat almost five times greater than the PBM group. The PBM group had significantly lower blood glucose levels 24 hours after the last session.

6. Clinical Applications of PBM for Inflammation

Amongst the many hundreds of reports of clinical applications of PBMT, we will highlight a few here, which seem to be especially relevant to inflammation, and inflammatory disorders.

6.1. Achilles tendinopathy

Bjordal et al in Norway carried out a randomized, placebo controlled trial of PBM (904 nm, 5.4 J per point, 20 mW/cm2) for activated Achilles tendinitis []. In addition to clinical assessment, they used microdialysis measurement of peritendinous prostaglandin E2 concentrations. Doppler ultrasonography measurements at baseline showed minor inflammation shown by increased intratendinous blood flow, and a measurable resistive index. PGE2 concentrations were significantly reduced with PBM vs placebo. The pressure pain threshold also increased significantly.

6.2. Thyroiditis

Chavantes and Chammas in Brazil have studied PBM for chronic autoimmune thyroiditis. An initial pilot trial [] used 10 applications of PBM (830 nm, 50 mW, 38–108 J/cm2), twice a week, using either the punctual technique (8 patients) or the sweep technique (7 patients). Patients required a lower dosage of levothyroxine, and showed an increased echogenicity by ultrasound. The next study [] was a randomized, placebo-controlled trial of 43 patients with a 9-month follow-up. In addition to improved thyroid function they found reduced autoimmunity evidenced by lower thyroid peroxidase antibodies (TPOAb), and thyroglobulin antibodies (TgAb). A third study [] used color Doppler ultrasound to show improved normal vascualrization in the thyroid parenchyma. Finally [] they showed a statistically significant increase in serum TGF-?1 levels 30 days post-intervention in the PBM group, thus confirming the anti-inflammatory effect. Recently a long-term follow up study of these thyroiditis patients (6 years later) was presented showing that PBM was safe in the long term and demonstrated lasting benefits [].

6.3. Muscles

PBM for muscles aims to benefit athletic performance and training, to reduce delayed onset muscle soreness (DOMS), as well as to ameliorate signs of muscle damage (creatine kinase) after intense or prolonged exercise. Moreover PBM can also be used to treat frank muscle damage caused by muscle strains or trauma. The International Olympic Committee and the World Anti-Doping Agency cannot ban light therapy for athletes considering (1) the intensity is similar to sunlight, and (2) there is no forensic test for light exposure. There have been several clinical trials carried out in Brazil in athletes such as elite runners [], volleyball players [] and rugby players []. Ferraresi et al conducted a case-controlled study in a pair of identical twins []. They used a flexible LED array (850 nm, 75 J, 15 sec) applied to both quadriceps femoris muscles (real to one twin and sham to the other) immediately after each strength training session (3 times/wk for 12 weeks) consisting of leg press and leg extension exercises with load of 80% and 50% of the 1-repetition maximum test, respectively. PBM increased the maximal load in exercise and reduced fatigue, creatine kinase, and visual analog scale (DOMS) compared to sham. Muscle biopsies were taken before and after the training program and showed that PBM decreased inflammatory markers such as interleukin 1? and muscle atrophy (myostatin). Protein synthesis (mammalian target of rapamycin) and oxidative stress defense (SOD2, mitochondrial superoxide dismutase) were up-regulated.

6.4. Psoriasis

Psoriasis is a chronic autoimmune skin disease. Psoriasis is characterized by the abnormally excessive and rapid growth of keratinocytes (instead of being replaced every 28–30 days as in normal skin, in psoriatic skin they are replaced every 3–5 days). This hyperproliferation is caused by an inflammatory cascade in the dermis involving dendritic cells, macrophages, and T cells secreting TNF-?, IL-1?, IL-6, IL-17, IL-22, and IL-36? []. PBM has been used for psoriasis because of its anti-inflammatory effects, which is a different approach from UV phototherapy which tends to kill circulating T-cells. Ablon [] tested PBM using LEDs (830 nm, 60 J/cm2 and 633 nm, 126 J/cm2) in two 20-min sessions over 4 or 5 weeks, with 48 h between sessions in 9 patients with chronic treatment-resistant psoriasis. Clearance rates at the end of the follow-up period ranged from 60% to 100%. Satisfaction was universally very high.

Choi et al [] tested PBM in case report of a patient with another inflammatory skin disease called acrodermatitis continua, who also had a 10-yr history of plaque psoriasis on her knees and elbows. As she was pregnant and not suited for pharmacological therapy, she received treatment with PBM (broad-band polarized light, 480–3,400 nm, 10 J/cm2). In two weeks (after only 4 treatments), the clinical resolution was impressive and no pustules were found. Topical methylprednisolone aceponate steroid cream was switched to a moisturizer, and she was treated twice or once a week with PBM until a healthy baby was delivered.

6.5. Arthritis

As can be seen from the animal studies section, arthritis is one of the most important clinical indications for PBM [,]. The two most common forms of arthritis are osteoarthritis (degenerative joint disease that mostly affects the fingers, knees, and hips) and rheumatoid arthritis (autoimmune joint inflammation that often affects the hands and feet). Osteoarthritis (OA) affects more than 3.8% of the population while rheumatoid arthritis (RA) affects about 0.24%. Both types have been successfully treated with PBM. Cochrane systematic reviews found for good evidence for its effectiveness in RA [], and some evidence in the case of OA []. Most clinical studies have used pain scales and range of movement scores to test the effectiveness, rather than measures of inflammation which are difficult to carry out in human subjects.

Barabas and coworkers [] made an attempt by testing PBM on ex vivo samples of synovial tissue surgically removed from patients receiving knee joint replacement. Synovial membrane samples received exposure to PBM (810 nm, 448 mW, 25 J/cm2, 1 cm2 area). PBM caused an increase in mitochondrial heat shock protein 1 60 kD, and decreases in calpain small subunit 1, tubulin alpha-1C, beta 2,vimentin variant 3, annexin A1, annexin A5, cofilin 1,transgelin, and collagen type VI alpha 2 chain precursor all significantly decreased compared to the control

6.6. Alopecia areata

Alopecia areata (AA) is one of the three common types of hair loss, the other two being androgenetic alopecia (AGA, male pattern baldness) and chemotherapy induced alopecia. AA is a common autoimmune disease resulting from damage caused to the hair follicles (HFs) by T cells. Evidence of autoantibodies to anagen stage HF structures is found in affected humans and experimental mouse models. Biopsy specimens from affected individuals demonstrate a characteristic peri- and intrafollicular inflammatory infiltrate around anagen-stage HFs consisting of activated CD4 and CD8 T lymphocytes []. PBM is an excellent treatment for hair loss in general and AGA in particular [,]. Yamazaki et al [] reported the use of the “Super-Lizer” delivering linear-polarized light between 600–1600 nm at a power of 1.26 W to the areas of hair loss on the scalp (4-s pulses delivered at 1-s intervals for 3 min every 1 or 2 weeks until hair growth was observed). Regrowth of vellus hairs was achieved on more than 50% ofthe involved areas in all 15 cases. The frequency of irradiation until regrowth ranged from one to 14 times and the duration of SL treatment was 2 weeks to 5 months.

7. Conclusion and Future Studies

The clinical applications of PBM have been increasing apace in recent years. The recent adoption of inexpensive large area LED arrays, that have replaced costly, small area laser beams with a risk of eye damage, has accelerated this increase in popularity. Advances in understanding of PBM mechanisms of action at a molecular and cellular level, have provided a scientific rationale for its use for multiple diseases. Many patients have become disillusioned with traditional pharmaceutical approaches to a range of chronic conditions, with their accompanying distressing side-effects and have turned to complementary and alternative medicine for more natural remedies. PBM has an almost complete lack of reported adverse effects, provided the parameters are understood at least at a basic level. The remarkable range of medical benefits provided by PBM, has led some to suggest that it may be “too good to be true”. However one of the most general benefits of PBM that has recently emerged, is its pronounced anti-inflammatory effects. While the exact cellular signaling pathways responsible for this anti-inflammatory action are not yet completely understood, it is becoming clear that both local and systemic mechanisms are operating. The local reduction of edema, and reductions in markers of oxidative stress and pro-inflammatory cytokines are well established. However there also appears to be a systemic effect whereby light delivered to the body, can positively benefit distant tissues and organs.

There is a lot of scope for further work on PBM and inflammation. The intriguing benefits of PBM on some autoimmune diseases, suggests that this area may present a fertile area for researchers. There may be some overlap between the ability of PBM to activate and mobilize stem cells and progenitor cells, and its anti-inflammatory action, considering that one of the main benefits of exogenous stem cell therapy has been found to be its anti-inflammatory effect. The versatile benefits of PBM on the brain and the central nervous system, encourages further study of its ability to reduce neuroinflammation. Chronic diseases of the modern age involving systemic inflammation such as type II diabetes, obesity, Alzheimer’s disease, cardiovascular disease and endothelial dysfunction are again worth investigating in the context of PBM.

?

Figure 7

Human clinical indications concentrating on anti-inflammatory effects. Autoimmune thyroiditis; muscle exercise and recovery; Achilles tendinopathy; knee arthritis; psoriasis; alopecia areata.

Acknowledgments

MRH was supported by US NIH grants R01AI050875 and R21AI121700.

Footnotes

Conflict of Interest: The author declares no conflict of interest in this paper.

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Free Radic Res. 2016 Mar 17:1-11. [Epub ahead of print]

Low-level laser therapy attenuates the acute inflammatory response induced by muscle traumatic injury.

Silveira PC1,2, Scheffer DD1, Glaser V1, Remor AP1, Pinho RA2, Aguiar Junior AS1, Latini A1.

Author information

  • 1a Laboratório de Bioenergética e Estresse Oxidativo – LABOX, Departamento de Bioquímica , Universidade Federal de Santa Catarina , Florianópolis , Brazil ;
  • 2b Laboratório de Fisiologia e Bioquímica do Exercício – LaFiBe , Universidade do Extremo Sul Catarinense , Criciúma , Brazil.

Abstract

The purpose of this work was to investigate the effect of early and long-term low-level laser therapy (LLLT) on oxidative stress and inflammatory biomarkers after acute-traumatic muscle injury in Wistar rats. Animals were randomly divided into the following four groups: control group (CG), muscle injury group (IG), CG + LLLT, and IG + LLLT: laser treatment with doses of 3 and 5 J/cm2. Muscle traumatic injury was induced by a single-impact blunt trauma in the rat gastrocnemius. Irradiation for 3 or 5 J/cm2 was initiated 2, 12, and 24 h after muscle trauma induction, and the treatment was continued for five consecutive days. All the oxidant markers investigated. namely thiobarbituric acid-reactive substance, carbonyl, superoxide dismutase, glutathione peroxidase, and catalase, were increased as soon as 2 h after muscle injury and remained increased up to 24 h. These alterations were prevented by LLLT at a 3 J/cm2 dose given 2 h after the trauma. Similarly, LLLT prevented the trauma-induced proinflammatory state characterized by IL-6 and IL-10. In parallel, trauma-induced reduction in BDNF and VEGF, vascular remodeling and fiber-proliferating markers, was prevented by laser irradiation. In order to test whether the preventive effect of LLLT was also reflected in muscle functionality, we tested the locomotor activity, by measuring distance traveled and the number of rearings in the open field test. LLLT was effective in recovering the normal locomotion, indicating that the irradiation induced biostimulatory effects that accelerated or resolved the acute inflammatory response as well as the oxidant state elicited by the muscle trauma.

Lasers Med Sci. 2016 Mar 16. [Epub ahead of print]

Low-level laser therapy modulates pro-inflammatory cytokines after partial tenotomy.

Da Ré Guerra F1, Vieira CP2, Oliveira LP2, Marques PP3, Dos Santos Almeida M4, Pimentel ER2.

Author information

  • 1Department of Anatomy, Institute of Biomedical Science, Federal University of Alfenas – UNIFAL-MG, 37130-000, Alfenas, MG, Brazil. dgflavia@yahoo.com.br.
  • 2Department of Structural and Functional Biology, Institute of Biology, CP 6109, University of Campinas – UNICAMP, 13083-970, Campinas, SP, Brazil.
  • 3Department of Biochemistry, Institute of Biomedical Science, Federal University of Alfenas – UNIFAL-MG, 37130-000, Alfenas, MG, Brazil.
  • 4Department of Anatomy, Institute of Biomedical Science, Federal University of Alfenas – UNIFAL-MG, 37130-000, Alfenas, MG, Brazil.

Abstract

Tendon injuries give rise to substantial morbidity, and current understanding of the mechanisms involved in tendon injury and repair is limited. This lesion remains a clinical issue because the injury site becomes a region with a high incidence of recurrent rupture and has drawn the attention of researchers. We already demonstrated that low-level laser therapy (LLLT) stimulates the synthesis and organization of collagen I, MMP-9, and MMP-2 and improved the gait recovery of the treated animals. The aim of this study was to evaluate the effects of LLLT in the nitric oxide and cytokines profile during the inflammatory and remodeling phases. Adult male rats were divided into the following groups: G1-intact, G2- injured, G3-injured + LLLT (4 J/cm2 continuous), G4-injured + LLLT (4 J/cm2-20 Hz-pulsed laser). According to the analysis, the animals were euthanized on different dates (1, 4, 8, or 15 days after injury). ELISA assay of TNF-a, IL-1B, IL-10, and TGF-B was performed. Western blotting of isoform of nitric oxide synthase (i-NOS) and nitric oxide dosage experiments was conducted. Our results showed that the pulsed LLLT seems to exert an anti-inflammatory effect over injured tendons, with reduction of the release of proinflammatory cytokines, such as TNF-a and the decrease in the i-NOS activity. Thanks to the pain reduction and the facilitation of movement, there was a stimulation in the TGF-B and IL-1B release. In conclusion, we believe that pulsed LLLT worked effectively as a therapy to reestablish the tendon integrity after rupture.

Lasers Med Sci. 2016 Jan;31(1):33-40. doi: 10.1007/s10103-015-1821-7. Epub 2015 Oct 27.

Effects of low-intensity non-coherent light therapy on the inflammatory process in the calcaneal tendon of ovariectomized rats.

Helrigle C1, de Carvalho Pd2,3, Casalechi HL4, Leal-Junior EC1,4, Fernandes GH1, Helrigel PA5, Rabelo RL5, de Oliveira Aleixo-Junior I4, Aimbire F6,Albertini R1,4.

Author information

  • 1Post-graduation Program in Biophotonics Applied to Health Sciences, Universidade Nove de Julho, UNINOVE, São Paulo, SP, Brazil.
  • 2Post-graduation Program in Biophotonics Applied to Health Sciences, Universidade Nove de Julho, UNINOVE, São Paulo, SP, Brazil. ptpaulo@terra.com.br.
  • 3Post-graduation Program in Rehabilitation Sciences, Universidade Nove de Julho, UNINOVE, Rua Vergueiro, 235, 01504-001, São Paulo, SP, Brazil. ptpaulo@terra.com.br.
  • 4Post-graduation Program in Rehabilitation Sciences, Universidade Nove de Julho, UNINOVE, Rua Vergueiro, 235, 01504-001, São Paulo, SP, Brazil.
  • 5Medical Veterinary Department, Federal University of Goiás-UFG, Jataí, GO, Brazil.
  • 6Institute of Science and Technology – ICT, Federal University of São Paulo-Unifesp, São José dos Campos, SP, Brazil.

Abstract

The aim of this experimental study was to investigate the effects of low-intensity light-emitting diode (LED) phototherapy on the inflammatory process in the calcaneal tendon of ovariectomized rats (OVX) through the involvement of the inflammatory mediators interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-?). Thirty-five female Wistar rats were divided into 4 groups: 3 groups of OVX rats totaling 30 rats (untreated OVX rats [OVX injury group], treated OVX rats [OVX LED group], and control OVX rats; subgroups existed based on the sampling times, which were 3, 7, and 14 days) and 1 group of non-OVX rats (not OVX; n?=?5). Tendon injury was induced by trauma using a 208-g mass placed at 20 cm from the right tendon of each animal with energy of 0.70 J. The animals were treated 12 h after tendonitis with LED therapy and every 48 h thereafter until euthanasia (at 3, 7, or 14 days). The tendons were dissected and stored in liquid nitrogen at -196 °C, thawed only at the time of immunoenzymatic testing (ELISA). Groups treated with LED showed a decrease in the number of pro-inflammatory cells, IL-6, and TNF-? (p <0.05), and an increase in IL-10 (p?<?0.05) when compared to the not OVX group (p?<?0.05). It was concluded that low-intensity LED treatment using the parameters and wavelength of 945 nm in the time periods studied reduced the release of IL-6 and TNF-? and increased the release of IL-10, thereby improving the inflammatory response in OVX rats.

Lasers Med Sci. 2015 Jan;30(1):153-8. doi: 10.1007/s10103-014-1636-y. Epub 2014 Jul 29.

Low-level laser therapy in IL-1B, COX-2, and PGE2 modulation in partially injured Achilles tendon.

de Jesus JF1, Spadacci-Morena DD, dos Anjos Rabelo ND, Pinfildi CE, Fukuda TY, Plapler H.
Author information
1Interdisciplinary Surgical Science Program, Universidade Federal de São Paulo-UNIFESP, São Paulo, SP, 04021-001, Brazil, juliofernandes85@gmail.com.
Abstract
This study evaluated IL-1B, COX-2, and PGE2 modulation in partially injured Achilles tendons treated with low-level laser therapy (LLLT). Sixty-five male Wistar rats were used. Sixty were submitted to a direct injury on Achilles tendon and then distributed into six groups: LASER 1 (a single LLLT application), LASER 3 (three LLLT applications), and LASER 7 (seven LLLT applications) and Sham 1, 3, and 7 (the same injury but LLLT applications were simulated). The five remaining animals were allocated at control group (no procedure performed). LLLT (780 nm) was applied with 70 mW of mean power and 17.5 J/cm(2) of fluency for 10 s, once a day. The tendons were surgically removed and assessed immunohistochemically for IL-1B, COX-2, and PGE2. In comparisons with control (IL-1B: 100.5?±?92.5 / COX-2: 180.1±97.1 / PGE2: 187.8±128.8) IL-1B exhibited (mean±SD) near-normal level (p>0.05) at LASER 3 (142.0±162.4). COX-2 and PGE2 exhibited near-normal levels (p>0.05) at LASER 3 (COX-2: 176.9±75.4 / PGE2: 297.2±259.6) and LASER 7 (COX-2: 259.2±190.4 / PGE2: 587.1±409.7). LLLT decreased Achilles tendon’s inflammatory process.
Lasers Med Sci. 2014 Jun 29. [Epub ahead of print]

Expression of mPGES-1 and IP mRNA is reduced by LLLT in both subplantar and brain tissues in the model of peripheral inflammation induced by carrageenan.

Chagas LR1, Silva JA Jr, de Almeida Pires J, Costa MS.

Author information

  • 1Instituto de Pesquisa e Desenvolvimento – IP&D, Universidade do Vale do Paraíba – UNIVAP, Av. Shishima Hifumi, 2911. Urbanova, CEP 12244-000, São José dos Campos, SP, Brazil.

Abstract

The increase in PGE2 production by microsomal PGE synthase-1 (mPGES-1) in CNS contributes to the severity of the inflammatory and pain responses in the model of edema formation and hyperalgesia induced by carrageenan. PGI2, alike to PGE2, plays an important role in the inflammation. Low-level laser therapy (LLLT) has been used in the treatment of inflammatory pathologies, reducing both pain and the acute inflammatory process. In this work, we studied the effect of LLLT on the expression of both mPGES-1 and IP messenger RNA (mRNA), in either subplantar or total brain tissues obtained from rats submitted to model of edema formation and hyperalgesia induced by carrageenan administration. The test sample consisted of 30 rats divided into five groups: A1 (control-saline), A2 (carrageenan-0.5 mg/paw), A3 (carrageenan-0.5 mg/paw?+?LLLT), A4 (carrageenan-1.0 mg/paw), and A5 (carrageenan-1.0 mg/paw?+?LLLT). The animals from groups A3 and A5 were irradiated 1 h after induction of inflammation by carrageenan injection. Continuous-wave red laser with wavelengths of 660 nm and dose of 7.5 J/cm2 was used. Six hours after carrageenan-induced inflammation, mPGES-1 and prostacyclin receptor (IP) mRNA expression were significantly increased both in subplantar and brain tissues. LLLT was able to reduce both mPGES-1 and IP mRNA expression in subplantar and brain tissues. We suggest that LLLT is able to reduce both inflammation and hyperalgesia observed in the model of edema formation and hyperalgesia induced by carrageenan, by a mechanism involving the decrease in the expression of both mPGES-1 and IP.

Lasers Med Sci.  2012 Nov 24. [Epub ahead of print]

Infrared low-level diode laser on inflammatory process modulation in mice: pro- and anti-inflammatory cytokines.

Fukuda TY, Tanji MM, Silva SR, Sato MN, Plapler H.

Source

Physical Therapy Sector, Irmandade da Santa Casa de Misericórdia de São Paulo (ISCMSP), São Paulo, São Paulo, Brazil, tfukuda10@yahoo.com.br.

Abstract

To evaluate the modulation of proinflammatory (interleukin-6, IL-6; tumor necrosis factor-a, TNF-a; and interferon-y, IFN-y) and anti-inflammatory cytokines (transforming growth factor-B1, TGF-B1) in the inflammation processes in vivo with low-level laser action, 50 isogenic mice were randomly distributed into three groups: control (no surgical procedure, n=10), sham (surgical procedure with three standard cutaneous incisions, followed by an abdominal muscle incision and suture, n=20), and laser (same procedure followed by laser exposure, n=20). The sham group was divided into three subgroups: sham I (euthanasia and evaluation, 36 h after surgical procedure), sham II (euthanasia and evaluation, 60 h after surgical procedure), and sham III (euthanasia and evaluation, 84 h after surgical procedure). The laser group was also divided in three subgroups: laser I (a single laser session, 12 h after surgery), laser II (two laser sessions, 12 and 36 h after surgery), and laser III (three laser sessions, 12, 36, and 60 h after surgery). All animals in the laser groups received three points per session of continuous infrared laser (wavelength of 780 nm, power of 20 mW, fluency of 10 J/cm(2), exposure time of 20 s per point, and energy of 0.4 J). After euthanasia, spleen mononuclear cells were isolated and cultured for 48 h. Concentrations of IL-6, TNF-a, IFN-y, and TGF-B1 were obtained by enzyme-linked immunosorbent assay method. There was a significant difference between the IL-6 and TNF-a concentrations in the 60-and 84-h evaluations when the laser and sham groups were compared to the control group (p<0.05), except for laser II in the TNF-a analysis (p>0.05). The IFN-y concentration analysis showed a significant difference only in sham II when compared to the control group (p<0.05). Thus, there was a modulatory effect of TNF-a and IFN-y in the laser group, particularly in the 60-h postoperative evaluation. There was no significant difference between the laser, sham, and control groups for TGF-B1 analysis (p>0.05). The low-level laser application decreased the TNF-a and IFN-y release in vivo of spleen mononuclear cells in mice, especially after two exposure sessions. However, there was no modulation of the IL-6 and TGF-B1 release.

Lasers Med Sci.  2012 Aug 31. [Epub ahead of print]

Low-level laser therapy improves the inflammatory profile of rats with heart failure.

Hentschke VS, Jaenisch RB, Schmeing LA, Cavinato PR, Xavier LL, Dal Lago P.

Source

Laboratório de Fisiologia, Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Porto Alegre, RS, Brazil.

Abstract

Following heart failure (HF), immune activation leads to an imbalance between pro-inflammatory and anti-inflammatory cytokines. Low-level laser therapy (LLLT) has been used as an anti-inflammatory treatment in several disease conditions. However, the effect of LLLT on the skeletal muscle of rats with HF remains unclear. The present report aimed to evaluate the influence of LLLT on the inflammatory profile of rats with HF. The left coronary artery was ligated to induce HF and a sham operation was performed in the control groups. Male Wistar rats (n=49) were assigned to one of six groups: placebo sham rats (P-Sham; n=8), LLLT at a dose of 3 J/cm(2) sham rats (3 J/cm(2)-Sham; n=8), LLLT at a dose of 21 J/cm(2) sham rats (21 J/cm(2)-Sham; n=8), placebo HF rats (P-HF; n=9), LLLT at a dose of 3 J/cm(2) HF rats (3 J/cm(2)-HF; n=8), and LLLT at a dose of 21 J/cm(2) HF rats (21 J/cm(2)-HF; n=8). Four weeks after myocardial infarction or sham surgery, rats were subjected to LLLT (InGaAlP 660 nm, spot size 0.035 cm(2), output power 20 mW, power density 0.571 W/cm(2), energy density 3 or 21 J/cm(2), exposure time 5.25 s and 36.75 s) on the right gastrocnemius for 10 consecutive days. LLLT reduced plasma IL-6 levels (61.3 %; P<0.01), TNF-/IL-10 (61.0 %; P<0.01) and IL-6/IL-10 ratios (77.3 %; P?<?0.001) and increased IL-10 levels (103 %; P<0.05) in the 21 J/cm(2)-HF group. Moreover, LLLT reduced the TNF-(20.1 % and 21.3 %; both P=<0.05) and IL-6 levels (54.3 % and 37.8 %; P?<0.01 and P<0.05, respectively) and the IL-6/IL-10 ratio (59.7 % and 42.2 %; P<?0.001 and P<0.05, respectively) and increased IL-10 levels (81.0 % and 85.1 %; both P<0.05) and the IL-10/TNF- ratio (171.5 % and 119.8 %; P<0.001 and P<0.05, respectively) in the gastrocnemius in the 3 J/cm(2)-HF and 21 J/cm(2)-HF groups. LLLT showed systemic and skeletal muscle anti-inflammatory effects in rats with HF.

Lasers Med Sci.  2012 Aug 23. [Epub ahead of print]

Prevention and treatment of mice paw edema by near-infrared low-level laser therapy on lymph nodes.

Meneguzzo DT, Lopes LA, Pallota R, Soares-Ferreira L, Lopes-Martins RA, Ribeiro MS.

Source

Center for Lasers and Applications, IPEN-CNEN/SP, Rua da Lagoa 541/15, Campinas, São Paulo, 13104-118, Brazil, daitm@uol.com.br

AbstractLow-level laser therapy (LLLT) has been demonstrated to modulate inflammatory processes and immunological responses. The aim of this work was to investigate the hypothesis that near infrared LLLT (830 nm) over lymph nodes may reduce paw edema and contribute to the modulation of inflammation. The edema was induced by carrageenan inoculation (CGN) into the plantar surface of 100 male mice left hind paw. Animals were divided into five groups: CGN (control), no treatment; Diclo, sodium diclofenac; Paw, LLLT on the paw; Ly, LLLT on the inguinal lymph nodes; and Paw+Ly, LLLT in both paw and lymph nodes, and subdivided according to moment of irradiation: A-1 h and 2 h before CGN, B-1 h and immediately before CGN, C-1 and 2 h after CGN, and D-3.5 and 4.5 h after CGN. The parameters used were: energy=1 J, fluence=35 J/cm(2), power=100 mW during 10 s. Paw volume was measured before and 1 to 6 h after CGN, and myeloperoxidase (MPO) activity was analyzed. Edema prevention was obtained by the irradiation of Paw+Ly at moment A and at Ly at moment B, inhibition of edema formation was achieved by either Paw or Ly at moment C, and edema treatment was obtained by Paw or Ly at moment D (p<0.05). MPO activity was significantly reduced on Paw at moment A, Paw and Ly on C, and in all irradiated groups on B and D. Our results suggest that LLLT was able to produce both anti-inflammatory and pro-inflammatory effects depending on to the site and moment of irradiation.

Lasers Med Sci.  2012 May;27(3):585-91. Epub 2011 Sep 27.

Antiinflammatory effect of low-level laser therapy on Staphylococcus epidermidis endophthalmitis in rabbits.

Ma WJ, Li XR, Li YX, Xue ZX, Yin HJ, Ma H.

Source

Tianjin Medical University Eye Centre, Tianjin, 300070, China.

Abstract

A rabbit model of endophthalmitis was established to evaluate the antiinflammatory effect of low-level laser therapy (LLLT) as an adjunct to treatment for Staphylococcus epidermidis endophthalmitis. Rabbits were randomly divided into three groups to receive intravitreal injections into their left eye: group A received 0.5 mg vancomycin (100 l), group B received 0.5 mg vancomycin + 0.2 mg dexamethasone (100 l), and group C received 0.5 mg vancomycin (100 l) and continuous wave semiconductor laser irradiation (10 mW, =632 nm) focused on the pupil. Slit lamp examination and B-mode ultrasonography were conducted to evaluate the symptoms of endophthalmitis. Polymorphonuclear cells and tumour necrosis factor alpha (TNF-) in aqueous fluid were measured at 0 h, and 1, 2, 3, 7 and 15 days. A histology test was conducted at 15 days. B-mode ultrasonography and histology revealed that groups B and C had less inflammation than group A at 15 days. Groups B and C had fewer polymorphonuclear cells and lower levels of TNF- in aqueous fluid than group A at 2, 3 and 7 days (P < 0.05). There was no significant difference between groups B and C (P > 0.05). There was no significant difference between groups A, B and C at 15 days (P > 0.05). As an adjunct to vancomycin therapy to treat S. epidermidis endophthalmitis, LLLT has an antiinflammatory effect similar to that of dexamethasone.

Lasers Med Sci.  2012 May;27(3):621-7. Epub 2011 Oct 16.

Low-level diode laser therapy reduces lipopolysaccharide (LPS)-induced bone cell inflammation.

Huang TH, Lu YC, Kao CT.

Source

Graduate School of Dentistry, Chung Shan Medical University Hospital, Chung Shan Medical University, 110, section 1, Chien Kuo N Road, Taichung City, 40201, Taiwan.

Abstract

In this study, the aim is to investigate the cytologic effects of inflammatory bone cells after in vitro low-level laser therapy (LLLT). A human osteosarcoma cell line (MG63) was cultured, infected with lipopolysaccharide (LPS) and exposed to low-level laser treatment at 5 or 10 J/cm(2) using a 920 nm diode laser. MG63 cell attachment was observed under a microscope, and cell viability was quantified by mitochondrial colorimetric assay (MTT). LPS-treated MG63 cells were irradiated with LLLT, and the inflammatory markers iNOS, TNF- and IL-1, were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The data were collected and analyzed by one-way analysis of variance (ANOVA); p<0.05 indicated a statistically significant difference. Low-level laser treatment on MG63 cells increased their ability to attach and survive. After irradiation, the expression levels of iNOS, TNF- and IL-1 in LPS-infected MG63 cells decreased over time (p<0.05). Conclusions: low-level diode laser treatment increased the MG63 cell proliferative ability and decreased the expression of inflammatory mediators in MG63 cells.

Lasers Med Sci.  2012 Jun 26. [Epub ahead of print]

Effect of the intravascular low energy laser illumination during percutaneous coronary intervention on the inflammatory process in vascular wall.

Derkacz A, Protasiewicz M, Por?ba R, Doroszko A, Andrzejak R.

Source

Department of Internal Medicine, Occupational Diseases and Hypertension, Wroclaw Medical University, Borowska 213 Street, 50-556, Wroclaw, Poland, arkadiusz.derkacz@am.wroc.pl

Abstract

The angioplasty procedure is associated with a release of numerous factors triggering the local inflammatory reaction in vascular wall and leading thus to the restenosis. In this study, we hypothesize that the low-energy laser irradiation may exert beneficial effect by limiting this process. A group of 101 subjects (75 men and 26 women, mean age: 59.1±10.3) treated with percutaneous coronary intervention (PCI), were recruited to this study. While 52 patients (40 men and 12 women) were subjected to the intravascular low-energy laser irradiation (808 nm) of dilated lesion during the PCI, the remaining patients (35 men and 14 women) constituted the control group. The levels of interleukin 1?, 6 and 10 (IL 1, IL 6 and IL 10) were measured immediately before the procedure, and then at the 6th, 12th hour as well as after 1 month following the PCI. Significantly lower levels of IL 1 and IL 6 in the irradiated group during each analysis after the procedure were observed. Moreover, significantly lower IL 10 level in irradiated group within 6 and 12 hours after PCI was observed. Irradiation of the lesion with low-energy laser radiation during the PCI procedure results in a decrease in the levels of pro-inflammatory IL 1 and IL 6 as well as in an increase in the levels of anti-inflammatory IL 10, which may result in decreased risk for restenosis.

Rev Bras Fisioter. 2011 Mar 18. pii: S1413-35552011005000005. [Epub ahead of print]

Inhibition of carrageenan-induced expression of tissue and plasma prekallikreins mRNA by low level laser therapy in a rat paw edema model.

Silva MP, Bortone F, Silva MP, Araújo TR, Costa MS, Silva Júnior JA.

Universidade Nove de Julho, São Paulo, SP, Brazil.

Abstract

BACKGROND: Low level laser therapy (LLLT) has been used clinically in order to treat inflammation, where tissue and plasma prekallikrein have crucial importance. Plasma prekallikrein (PPK) is synthesized by the hepatocytes and secreted into the bloodstream, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Tissue prekallikrein is associated with important disease states (including cancer, inflammation, and neurodegeneration) and has been utilized or proposed as clinically important biomarker or therapeutic target of interest.

OBJECTIVE: To evaluate if LLLT modulates tissue and plasma prekallikreins mRNA expression in the carrageenan-induced rat paw edema.

METHODS: Experimental groups were assigned as followed: A1 (Control-saline), A2 (Carrageenan-only), A3 (laser 660nm only) and A4 (Carrageenan + laser 660nm). Edema was measured by a plethysmometer. Subplantar tissue was collected for the quantification of prekallikreins mRNA by Real time-Polymerase Chain Reaction.

RESULTS: A significantly decrease in the edema was observed after laser irradiation. Expression of prekallikreins increased after carrageenan injection. Tissue and plasma prekallikrein mRNA expression significantly decreased after LLLT’s 660nm wavelength.

CONCLUSION: These results suggest that expression of tissue and plasma prekallikreins is modulated by LLLT, which can be used in clinical practice due to its anti-inflammatory effects.

Photomed Laser Surg. 2010 Dec;28(6):763-71.

Low-Level Laser Therapy Associated to N-Acetylcysteine Lowers Macrophage Inflammatory Protein-2 (MIP-2) mRNA Expression and Generation of Intracellular Reactive Oxygen Species in Alveolar Macrophages.

de Lima FM, Villaverde AB, Albertini R, de Oliveira AP, Neto HC, Aimbire F.

1 Institute of Research and Development , IP&D, UNIVAP, São José dos Campos, São Paulo, Brazil .

Abstract

Abstract Objective: The aim of this work was to investigate the low-level laser therapy (LLLT) effect on alveolar macrophages (AM) activated by oxidative stress and lipopolysaccharide (LPS).

Background data: LLLT has been reported to actuate positively relieving the late and early symptoms of airway and lung inflammation. It is not known if the increased MIP-2?mRNA expression and intracellular reactive oxygen species (ROS) generation observed in acute lung inflammation (ALI) can be influenced by LLLT.

Materials and Methods: Rat AM cell line (AMJ2-C11) was cultured with LPS or H(2)O(2) and laser irradiated. MIP-2?mRNA and ROS production in the AM were evaluated by Real Time-PCR and the 2?,7?-dichlorofluorescin diacetate (DCFH-DA) respectively. The NF-?B protein in the AM was measured by the enzyme linked immunoassay method. To investigate the antioxidant effect of laser, the AM were prebathed with N-acetylcysteine (NAC) and then irradiated with laser. LLLT was also studied in the presence of an inhibitor of NF-?B (BMS 205820). In addition, the effect of LLLT on NF-?B protein was investigated.

Results: LLLT attenuated the MIP-2?mRNA expression and intracellular ROS generation after LPS or H(2)O(2). When the AM were pretreated with NAC, the laser effect was potentiated. BMS 205820 suppresses the effect of LLLT on MIP-2?mRNA expression and ROS generation, stimulated by LPS or H(2)O(2). On NF-?B transcription factor, both the LLLT and NAC reduced this protein in the AM exposed to LPS or H(2)O(2). The synergistic effect between LLLT and NAC on the reduction the NF-?B was also evidenced.

Conclusion: Results indicate that there is a synergistic action of LLLT with NAC on MIP-2?mRNA expression from LPS- or H(2)O(2)-stimulated AM, and that both ROS intracellular generation and NF-kB signaling seem to be involved.

Physical Therapy Reviews, Volume 15, Number 4, August 2010 , pp. 286-293(8)

The anti-inflammatory mechanism of low level laser therapy and its relevance for clinical use in physiotherapy

Authors: Bjordal, Jan Magnus1; Lopes-Martins, Rodrigo Alvaro Brand?o2; Joensen, Jon3; Iversen, Vegard Vereide4

Source: Physical Therapy Reviews, Volume 15, Number 4

Abstract:

Background: Low level laser therapy (LLLT) is a modality that has been used by physiotherapists for more than two decades. Clinical use has largely relied on empirical data, but new evidence suggests that LLLT can trigger specific photobiological mechanisms.

Objective: To review possible therapeutic windows for LLLT in inflammatory reactions.

Methods: Systematic review of LLLT in studies with cell cultures and animals where inflammation is induced. Skin wound studies were excluded unless they measured the influence of drugs on LLLT effects, or made a direct comparison of LLLT and drugs in inflammation.

Results: We identified 1 review, 34 cell studies, 54 animal studies and 106 skin incision studies potentially eligible for analysis. Eleven cell studies and 27 animals studies met all our inclusion criteria, and another six animal studies met our inclusion criteria for drug comparisons and LLLT interactions. There is strong evidence of an anti-inflammatory effect from LLLT, which is consistent across all 12 tested laboratory models and phases of inflammation and wavelengths between 633 and 904 nm. The magnitude of the antiinflammatory effect is not significantly different from that of non-steroidal anti-inflammatory drugs (NSAIDs), but it is slightly less than glucocorticoid steroids. There is moderate evidence that concomitant use of glucocorticoid steroid has a negative effect on LLLT mechanisms and should be avoided.

Conclusion: Red and near infrared LLLT administered with mean laser output of 2.5–100 mW, irradiation times of 16–600 s and doses of 0.6–9.6 J reduces inflammation significantly, and is equally effective as NSAIDs in animal laboratory studies. Scattered evidence from human studies have found similar antiinflammatory effects of LLLT, suggesting that this mechanism may be responsible for many of the significant effects reported in clinical LLLT studies.

Lasers Med Sci. 2010 Aug 25. [Epub ahead of print]

Low-level laser therapy (LLLT; 780 nm) acts differently on mRNA expression of anti- and pro-inflammatory mediators in an experimental model of collagenase-induced tendinitis in rat.

Pires D<>, Xavier M<>, Araújo T<>, Silva JA Jr<>, Aimbire F<>, Albertini R<>.

Institute of Research and Development, Av. Shishima Hifumi, 2911, Urbanova, CEP, 12.244-000, São José dos Campos, SP, Brazil.

Abstract

Low-level laser therapy (LLLT) has been found to produce anti-inflammatory effects in a variety of disorders. Tendinopathies are directly related to unbalance in expression of pro- and anti-inflammatory cytokines which are responsible by degeneration process of tendinocytes. In the current study, we decided to investigate if LLLT could reduce mRNA expression for TNF-alpha, IL-1beta, IL-6, TGF-beta cytokines, and COX-2 enzyme. Forty-two male Wistar rats were divided randomly in seven groups, and tendinitis was induced with a collagenase intratendinea injection. The mRNA expression was evaluated by real-time PCR in 7th and 14th days after tendinitis. LLLT irradiation with wavelength of 780 nm required for 75 s with a dose of 7.7 J/cm(2) was administered in distinct moments: 12 h and 7 days post tendinitis. At the 12 h after tendinitis, the animals were irradiated once in intercalate days until the 7th or 14th day in and them the animals were killed, respectively. In other series, 7 days after tendinitis, the animals were irradiated once in intercalated days until the 14th day and then the animals were killed. LLLT in both acute and chronic phases decreased IL-6, COX-2, and TGF-beta expression after tendinitis, respectively, when compared to tendinitis groups: IL-6, COX-2, and TGF-beta. The LLLT not altered IL-1beta expression in any time, but reduced the TNF-alpha expression; however, only at chronic phase. We conclude that LLLT administered with this protocol reduces one of features of tendinopathies that is mRNA expression for pro-inflammatory mediators.

Lasers Surg Med. 2010 Aug;42(6):553-8.

Anti-inflammatory effects of low-level light emitting diode therapy on Achilles tendinitis in rats.

Xavier M<>, David DR<>, de Souza RA<>, Arrieiro AN<>, Miranda H<>, Santana ET<>, Silva JA Jr<>, Salgado MA<>, Aimbire F<>, Albertini R<>.

Institute of Research and Development, IP&D, Vale do Paraiba University, UNIVAP, Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, São Paulo, Brazil.

Abstract

BACKGROUND AND OBJECTIVES: The present study investigated the effects of low-level light emitting diode (LED) therapy (880 +/- 10 nm) on inflammatory process in a experimental model of Achilles tendinitis induced by collagenase.

STUDY DESIGN/MATERIALS AND METHODS: Fifty-six male Wistar were separated into seven groups (n = 8), three groups in the experimental period of 7 days and four groups in the experimental period of 14 days, the control group (CONT), tendinitis group (TEND), LED therapy group (LEDT) for both experimental periods, and LED therapy group 7th to 14th day (LEDT delay) for 14 days experimental period. The LED parameters was 22 mW CW of optical output power, distributed in an irradiation area of 0.5 cm(2), with an irradiation time of 170 seconds, the applied energy density was 7.5 J/cm(2) in contact. The therapy was initiated 12 hours after the tendinitis induction, with a 48-hour interval between the irradiations. The histological analysis and inflammatory mediators were quantified.

RESULTS: Our results showed that LED decreases the inflammatory cells influx and mRNA expression to IL-1 beta, IL-6, tumor necrosis factor-alpha (TNF-alpha) in both phase, and cyclooxygenase-2 (COX-2) just in initial phase (P < 0.05).

CONCLUSION: Our results suggest that the anti-inflammatory therapy with low-power LED (880 nm) enhanced the tissue response in all groups. We can conclude that the LED was able to reduce signs of inflammation in collagenase-induced tendinitis in rats by reducing the number of inflammatory cells and decrease mRNA expression of cytokines.

Lasers Surg Med. 2010 Aug;42(6):577-83.

Evaluation of inflammatory biomarkers associated with oxidative stress and histological assessment of low-level laser therapy in experimental myopathy.

Servetto N<>, Cremonezzi D<>, Simes JC<>, Moya M<>, Soriano F<>, Palma JA<>, Campana VR<>.

Cátedra de Física Biomédica, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Santa Rosa 1085, (5000) Córdoba, Argentina.

Abstract

The objective of the present work was to study the effect of helium-neon (He-Ne) and gallium-arsenide (Ga-As) laser upon inflammatory biomarkers associated with oxidative stress: fibrinogen, nitric oxide (NO), L-citrulline, and superoxide dismutase (SOD). These were evaluated through histological assessment, in rats with experimental myopathy.

MATERIALS AND METHODS: The groups studied were: (A) control, (B) injured, (C) injured and treated with He-Ne laser, (D) injured and treated with Ga-As laser, (E) irradiated with He-Ne; and (F) irradiated with Ga-As laser. Myopathy was induced by injecting 0.05 mg/rat/day of adrenaline in the left posterior limb muscle at the same point on 5 consecutive days, in groups B, C, and D. Low-level laser therapy (LLLT) was applied with 9.5 J/cm(2) daily for 7 consecutive days with each laser. The determination of the biomarkers was made by spectrophotometry. The muscles (5/8, single blinded) were stained with Gomori Trichrome and examined by optic microscopy. The quantitative variables were statistically analyzed by the Fisher’s test and categorical data by the Axionvision 4.8 program. Pearson’s chi-squared test was applied, setting significant difference at P < 0.05 for all cases.

RESULTS: In group B, the biomarkers were significantly increased compared to the other groups (P < 0.001), except for NO which in group B decreased significantly (P < 0.001). In group B, there was a higher inflammatory infiltration level (80.67%) in relation to destroyed fibers.

CONCLUSIONS: LLLT caused significant changes in inflammatory biomarkers and oxidative stress: decreased levels of fibrinogen, L-citrulline and SOD as opposed to the increase of NO in rats with experimental myopathies and significant muscle recovery.

Med Pregl. 2010 Mar-Apr;63(3-4):188-93.

Stimulation of mucoperiostal slice epithelization by small power laser after the primary plastic of oroantral communication.

[Article in Serbian]

Jovanovi? G, Buri? N, Tijanic M.

Klinika za stomatologiju, Medicinski fakultet, Nis.

Abstract

INTRODUCTION: The oroantral communication is a pathologic communication between the oral cavity and maxillary sinus. It originates with extraction of the upper lateral teeth. Primary plastics communication, which is more extensive than 5 mm has been unsuccessful in 16%. Small power lasers have positive reaction on wounds healing. The aim of this work was to determine the lasers effects on slice epithelization after the plastics more extensive than 5 mm.

MATERIAL AND METHODS: The experimental research was done on dogs with extraction of the upper second premolars on both sides, and formed oroantral communication having diameter of 10 mm. After the plastics of sinus, the left side slices were exposed to radiation for 7 days, and the opposite slices healed spontaneously. Eight points (8x1J) were treated for 100s by GaAlAs laser, power 10 mW and wavelength 670 nm. Biopses of the slices connections were taken on the 14th day to be laboratory treated and examined microscopially. The study included 36 examinees with communication diameter of 10 mm and performed plastics of sinuses. In half of the examinees wounds were exposed to radiation for 7 days, and in other examinees they healed spontaneously. The results were analyzed on the fourteenth day according to the scale: (1) complete healing, without dehiscention; (2) incomplete healing, with minimal dehiscention; (3) communication did not heal, with partial dehiscention; (4) open communication, with significant dehiscention.

RESULTS AND DISCUSSION: The microscopic analysis shows that no wounds exposed to radiation were overcast with mucous membrane which had smooth sub epithelia chronic inflammation and inflammable infiltrate, and strong fibroplasias and granulations. Wounds exposed to radiation had mucous membrane without any signals of inflammation. Laser radiation causes anti-inflammatory reaction, i.e., it provokes reduction of exudation, alteration and proliferation, it blocks cyclo- and lipo-oxygenation by delaying the synthesis of prostaglandin, stimulates neutrophyll, macrophage and lissome activity and it activates the function of immune complex T and B lymphocytes, so this difference could be primary referred to the action of laser. Our clinical study shows that complete healing of oroantral communication was recorded in 88.8% of the examinees who were exposed to radiation in relation to 50%, of those who did not receive radiation therapy which is statistically much higher percentage (chi2 test < 0.05). The surgery was repeated in 5.6% of those who had received radiation therapy and in 16.7% of those who had not been exposed to radiation. Laser radiation stimulates changing of ADP in ATP and it accelerates cells metabolism, it increases microcirculation and accelerates substance exchange of cells, it increases DNK and RNK synthesis and stimulates cells division, which cause quicker regeneration of epithelia, i.e., it accelerates the process of wound healing.

CONCLUSION: It can be concluded that small power laser can be used successfully as additional method of treatment, after closing of oroantral communication surgically.

Photomed Laser Surg. 2010 Feb;28(1):125-9.

 

Helium-neon laser reduces the inflammatory process of arthritis.

 

Rubio CR<>, Cremonezzi D<>, Moya M<>, Soriano F<>, Palma J<>, Campana V<>.

Cátedra de Física Biomédica, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Abstract

OBJECTIVE: A histological study of the anti-inflammatory effect of helium-neon laser in models of arthropathies induced by hydroxyapatite and calcium pyrophosphate in rats.

BACKGROUND: Crystal deposition diseases are inflammatory pathologies induced by cellular reaction to the deposit of crystals in the joints.

METHODS: Fifty-six Suquia strain rats were distributed in seven groups. Two mg of each crystal diluted in 0.05 ml physiologic solution were injected six times in each back limb joint, during two weeks on alternate days. Eight J/cm(2) were applied daily to the crystal-injected joints on five consecutive days. The joints were cut and put in 10% formaldehyde, stained with hematoxylin-eosin and observed by light microscopy. The percentage of area with inflammatory infiltrates was determined in five optical microscopy photographs (100X) for each group and analyzed using the Axionvision 4.6 program. A Pearson’s Chi Squared test was applied, with significance level set at p < 0.05.

RESULTS: Both crystals produced an inflammatory process in the osteoarticular structures, consisting of predominantly mononuclear infiltration, fibrosis, and granulomas of foreign body-type giant cells containing phagocytosed remains of crystals. In the arthritic joints treated with laser, a marked decrease (p < 0.0001) was found in the percentage of area with inflammatory infiltrates, although the granulomas remained in a less ostensible form, with adipose tissue cells, fibrosis bands with light residual inflammation, and an absence of or very few crystals. Laser alone or physiologic solution injection did not produce histological changes.

CONCLUSIONS: Helium-neon laser reduced the intensity of the inflammatory process in the arthritis model induced by hydroxyapatite and calcium pyrophosphate crystals.

Photomed Laser Surg. 2009 Feb;27(1):79-84.

Inflammatory and oxidative stress markers in experimental crystalopathy: their modification by photostimulation.

Rubio CR<>, Simes JC<>, Moya M<>, Soriano F<>, Palma JA<>, Campana V<>.

Cátedra de Física Biomédica, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Abstract

Crystalopathies are inflammatory pathologies caused by cellular reactions to the deposition of crystals in the joints. The anti-inflammatory effect of the helium-neon (He-Ne) laser and that of the nonsteroidal anti-inflammatory drugs (NSAIDs) diclofenac, meloxicam, celecoxib, and rofecoxib was studied in acute and chronic arthritis produced by hydroxyapatite and calcium pyrophosphate in rats. The presence of the markers fibrinogen, L-citrulline, nitric oxide, and nitrotyrosine was determined. Crystals were injected into the posterior limb joints of the rats. A dose of 8 J/cm(2) of energy from an He-Ne laser was applied for 3 d in some groups and for 5 d in other groups. The levels of some of the biomarkers were determined by spectrophotometry, and that of nitrotyrosine was determined by ELISA. For statistical analysis, Fisher’s exact test was used, and p +/- 0.05 was considered significant. In arthritic rats, the fibrinogen, L-citrulline, nitric oxide, and nitrotyrosine levels increased in comparison to controls and to the laser-treated arthritic groups (p +/- 0.001), (p +/- 0.001), (p +/- 0.02), and (p +/- 0.01), respectively. When comparing fibrinogen from arthritic rats with disease induced by hydroxyapatite with undiseased and arthritic rats treated with NSAIDs, the He-Ne laser decreased levels to values similar to those seen in controls (p +/- 0.01). Inflammatory and oxidative stress markers in experimental crystalopathy are positively modified by photobiostimulation.

Lasers Surg Med. 2009 Jan;41(1):68-74.

Low level laser therapy (LLLT): attenuation of cholinergic hyperreactivity, beta(2)-adrenergic hyporesponsiveness and TNF-alpha mRNA expression in rat bronchi segments in E. coli lipopolysaccharide-induced airway inflammation by a NF-kappaB dependent mechanism.

Mafra de Lima F<>, Costa MS<>, Albertini R<>, Silva JA Jr<>, Aimbire F<>.

Institute of Research and Development (IP&D), São Paulo, Brazil.

BACKGROUND AND OBJECTIVES: It is unknown if the decreased ability to relax airways smooth muscles in asthma and other inflammatory disorders, such as acute respiratory distress syndrome (ARDS), can be influenced by low level laser therapy (LLLT) irradiation. In this context, the present work was developed in order to investigate if LLLT could reduce dysfunction in inflamed bronchi smooth muscles (BSM) in rats.

STUDY DESIGN/MATERIALS AND METHODS: A controlled ex vivo study was developed where bronchi from Wistar rat were dissected and mounted in an organ bath apparatus with or without a TNF-alpha.

RESULTS: LLLT administered perpendicularly to a point in the middle of the dissected bronchi with a wavelength of 655 nm and a dose of 2.6 J/cm(2), partially decreased BSM hyperreactivity to cholinergic agonist, restored BSM relaxation to isoproterenol and reduced the TNF-alpha mRNA expression. An NF-kappaB antagonist (BMS205820) blocked the LLLT effect on dysfunction in inflamed BSM.

CONCLUSION: The results obtained in this work indicate that the LLLT effect on alterations in responsiveness of airway smooth muscles observed in TNF-alpha-induced experimental acute lung inflammation seems to be dependent of NF-kappaB activation.

Lasers Surg Med.   2008 Sep;40(7):500-8.

Anti-Inflammatory effects of low-level laser therapy (660 nm) in the early phase in carrageenan-induced pleurisy in rat.

Boschi ES, Leite CE, Saciura VC, Caberlon E, Lunardelli A, Bitencourt S, Melo DA, Oliveira JR.

Source

Laboratório de Pesquisa em Biofísica Celular e Inflamação, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681 prédio 12C sala 263, CEP 90619-900 Porto Alegre, RS, Brazil.

Abstract

BACKGROUND AND OBJECTIVE:

In the classic model of pleurisy there is little evidence about the anti-inflammatory effects of low-level laser therapy (LLLT) as well the dosage characteristics, such as wavelength, total energy, number and pattern of treatment. In this study we investigated the potential effects of LLLT on modulating the pro-inflammatory and anti-inflammatory mediators of acute inflammation in a rat pleurisy model.

STUDY DESIGN/MATERIALS AND METHODS:

A sample of 48 female Wistar rats were divided into control and experiential groups. An inflammation was induced by carrageenan (0.2 ml) injected into the pleural cavity. At 1, 2, and 3 hours after induction a continuous wave (20 mW) diode laser of the InGaAlP (660 nm) type was used in the four laser groups with different doses and treatment patterns. One group received a single dose of 2.1 J and the other three groups received a total energy of 0.9, 2.1, and 4.2 J. Four hours later the exudate volume, total and differential leukocytes, protein concentration, NO, IL-6, IL-10, TNF-alpha, and MCP-1 were measured from the aspirated liquid.

RESULTS:

All the treatment patterns and quantity of energy studied show significant reduction of the exudate volume (P<0.05). Using energy of 0.9 J only NO, IL-6, MCP-1 and IL-10 are significantly reduced (P<0.05). On the other hand, higher energies (2.1 and 4.2 J) significantly reduce all variables independently of the treatment pattern. The neutrophil migration has a straight correlation with the TNF-alpha (r = 0.551) and NO (r = 0.549) concentration.

CONCLUSIONS:

LLLT-660 nm induced an anti-inflammatory effect characterized by inhibition of either total or differential leukocyte influx, exudation, total protein, NO, IL-6, MCP-1, IL-10, and TNF-alpha, in a dose-dependent manner. Under these conditions, laser treatment with 2.1 J was more effective than 0.9 and 4.2 J.

Photomed Laser Surg. 2008 Feb;26(1):19-24.

Cytokine mRNA expression is decreased in the subplantar muscle of rat paw subjected to carrageenan-induced inflammation after low-level laser therapy.

Albertini R, Villaverde AB, Aimbire F, Bjordal J, Brugnera A, Mittmann J, Silva JA, Costa M.

Instituto de Pesquisa and Desenvolvimento (IP&D), Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, SP, Brazil.

Abstract

OBJECTIVE: The objective of this work was to investigate the anti-inflammatory effects of low-level laser therapy, applied at different wavelengths (660 and 684 nm), on cytokine mRNA expression after carrageenan-induced acute inflammation in rat paw.

BACKGROUND DATA: Low-level laser therapy (LLLT) has been observed to reduce pain in inflammatory disorders. However, little is known about the mechanisms behind this effect or whether it is wavelength-specific.

MATERIALS AND METHODS: The test sample consisted of 32 rats divided into four groups: A(1) (control-saline), A(2) (carrageenan-only), A(3) (carrageenan + 660 nm laser therapy), and A(4) (carrageenan + 684 nm laser therapy). The animals from groups A(3) and A(4) were irradiated 1 h after induction of inflammation by carrageenan injection. Continuous-wave red lasers with wavelengths of 660 and 684 nm and dose of 7.5 J/cm(2) were used.

RESULTS: Both the 660 nm and 684 nm laser groups had 30%-40% lower mRNA expression for cytokines TNF-alpha, IL-1beta, and IL-6 in the paw muscle tissue than the carrageenan-only control group. Cytokine measurements were made 3 h after laser irradiation of the paw muscle, and all cytokine differences between the carrageenan-only control group and the LLLT groups were statistically significant (p < 0.001).

CONCLUSIONS: LLLT at the 660-nm and 684-nm wavelengths administered to inflamed rat paw tissue at a dose of 7.5 J/cm(2) reduce cytokine mRNA expression levels within 3 h in the laser-irradiated tissue.

Lasers Surg Med. 2008 Jul;40(5):371-8.

Irradiation with 780 nm diode laser attenuates inflammatory cytokines but upregulates nitric oxide in lipopolysaccharide-stimulated macrophages: implications for the prevention of aneurysm progression.

Gavish L<>, Perez LS<>, Reissman P<>, Gertz SD<>.

Department of Anatomy and Cell Biology, The Hebrew University, Hadassah Medical School, Jerusalem 91120, Israel. lilachg@ekmd.huji.ac.il<>

Abstract

BACKGROUND AND OBJECTIVES: Low level laser irradiation (LLLI) has been shown to reduce inflammation in a variety of clinical situations. We have shown that LLLI (780 nm) increases aortic smooth muscle cell proliferation and matrix protein secretion and modulates activity and expression of matrix metalloproteinases. Inflammation is a major component of arteriosclerotic diseases including aneurysm. Macrophage recruitment and secretion of pro-inflammatory cytokines and the vasodilator, nitric oxide (NO), are central to most immune responses in the arterial wall. The present study was designed to determine the effect of LLLI on cytokine gene expression and secretion as well as gene expression of inducible nitric oxide synthase (iNOS) and NO production in lipopolysaccharide (LPS)-stimulated macrophages.

STUDY DESIGN/MATERIALS AND METHODS: Murine monocyte/macrophages (RAW 264.7) were irradiated with a 780 nm diode laser (2 mW/cm(2), 2.2 J/cm(2)) during stimulation with LPS (0, 0.1, and 1 microg/ml). Gene expression of chemokines, cytokines, and iNOS were assessed by RT-PCR. Secretion of interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 and NO were assessed by ELISA and the Griess reaction, respectively.

RESULTS: LLLI reduced gene expression of MCP-1, IL-1alpha, IL-10 (P<0.01), IL-1beta, and IL-6 (P<0.05) when cells were stimulated by 1 microg/ml LPS. LLLI reduced LPS-induced secretion of MCP-1 over non-irradiated cells by 17+/-5% and 13+/-5% at 12 hours (0.1 and 1 microg/ml LPS; P<0.01 and P=0.05, respectively), and reduced IL-1beta by 22+/-5% and 25+/-9% at 24 hours (0.1 and 1 microg/ml LPS, P=0.01 and P=0.06, respectively). However, LLLI increased NO secretion after 12 hours (LLLI vs. Control: without LPS, 1.72+/-0.37 vs. 0.95+/-0.4 microM, P<0.05; 0.1 microg/ml LPS, 7.46+/-1.62 vs. 4.44+/-1.73 microM, P=0.06; 1 microg/ml LPS, 10.91+/-3.53 vs. 6.88+/-1.52 microM, P<0.05).

CONCLUSIONS: These properties of LLLI, with its effects on smooth muscle cells reported previously, may be of profound therapeutic relevance for arterial diseases such as aneurysm where inflammatory processes and weakening of the matrix structure of the arterial wall are major pathologic components.

Inflammation. 2008 Jun;31(3):189-97. Epub 2008 Apr 18.

Low level laser therapy (LLLT) decreases pulmonary microvascular leakage, neutrophil influx and IL-1beta levels in airway and lung from rat subjected to LPS-induced inflammation.

Aimbire F<>, Ligeiro de Oliveira AP<>, Albertini R<>, Corrêa JC<>, Ladeira de Campos CB<>, Lyon JP<>, Silva JA Jr<>, Costa MS<>.

Instituto de Pesquisa & Desenvolvimento-IP&D, Universidade do Vale do Paraíba-UNIVAP, Av. Shishima Hifumi, 2911, CEP: 12244-000, São José dos Campos, São Paulo, Brazil. aimbire@univap.br

BACKGROUND AND OBJECTIVE: Low level laser therapy (LLLT) is a known anti-inflammatory therapy. Herein we studied the effect of LLLT on lung permeability and the IL-1beta level in LPS-induced pulmonary inflammation.

STUDY DESIGN/METHODOLOGY: Rats were divided into 12 groups (n = 7 for each group). Lung permeability was measured by quantifying extravasated albumin concentration in lung homogenate, inflammatory cells influx was determined by myeloperoxidase activity, IL-1beta in BAL was determined by ELISA and IL-1beta mRNA expression in trachea was evaluated by RT-PCR. The rats were irradiated on the skin over the upper bronchus at the site of tracheotomy after LPS.

RESULTS: LLLT attenuated lung permeability. In addition, there was reduced neutrophil influx, myeloperoxidase activity and both IL-1beta in BAL and IL-1beta mRNA expression in trachea obtained from animals subjected to LPS-induced inflammation.

CONCLUSION: LLLT reduced the lung permeability by a mechanism in which the IL-1beta seems to have an important role.

Lasers Surg Med. 2008 Jul;40(5):371-8.

Irradiation with 780 nm diode laser attenuates inflammatory cytokines but upregulates nitric oxide in lipo-polysaccharide-stimulated macrophages: implications for the prevention of aneurysm progression.

Gavish L, Perez LS, Reissman P, Gertz SD.

Department of Anatomy and Cell Biology, The Hebrew University, Hadassah Medical School, Jerusalem 91120, Israel. lilachg@ekmd.huji.ac.il

BACKGROUND AND OBJECTIVES: Low level laser irradiation (LLLI) has been shown to reduce inflammation in a variety of clinical situations. We have shown that LLLI (780 nm) increases aortic smooth muscle cell proliferation and matrix protein secretion and modulates activity and expression of matrix metalloproteinases. Inflammation is a major component of arteriosclerotic diseases including aneurysm. Macrophage recruitment and secretion of pro-inflammatory cytokines and the vasodilator, nitric oxide (NO), are central to most immune responses in the arterial wall. The present study was designed to determine the effect of LLLI on cytokine gene expression and secretion as well as gene expression of inducible nitric oxide synthase (iNOS) and NO production in lipopolysaccharide (LPS)-stimulated macrophages.

STUDY DESIGN/MATERIALS AND METHODS: Murine monocyte/macrophages (RAW 264.7) were irradiated with a 780 nm diode laser (2 mW/cm(2), 2.2 J/cm(2)) during stimulation with LPS (0, 0.1, and 1 microg/ml). Gene expression of chemokines, cytokines, and iNOS were assessed by RT-PCR. Secretion of interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 and NO were assessed by ELISA and the Griess reaction, respectively.

RESULTS: LLLI reduced gene expression of MCP-1, IL-1alpha, IL-10 (P<0.01), IL-1beta, and IL-6 (P<0.05) when cells were stimulated by 1 microg/ml LPS. LLLI reduced LPS-induced secretion of MCP-1 over non-irradiated cells by 17+/-5% and 13+/-5% at 12 hours (0.1 and 1 microg/ml LPS; P<0.01 and P=0.05, respectively), and reduced IL-1beta by 22+/-5% and 25+/-9% at 24 hours (0.1 and 1 microg/ml LPS, P=0.01 and P=0.06, respectively). However, LLLI increased NO secretion after 12 hours (LLLI vs. Control: without LPS, 1.72+/-0.37 vs. 0.95+/-0.4 microM, P<0.05; 0.1 microg/ml LPS, 7.46+/-1.62 vs. 4.44+/-1.73 microM, P=0.06; 1 microg/ml LPS, 10.91+/-3.53 vs. 6.88+/-1.52 microM, P<0.05).

CONCLUSIONS: These properties of LLLI, with its effects on smooth muscle cells reported previously, may be of profound therapeutic relevance for arterial diseases such as aneurysm where inflammatory processes and weakening of the matrix structure of the arterial wall are major pathologic components.

Bioelectromagnetics. 2008 Apr;29(3):197-206.

Anti-inflammatory effects of low-intensity extremely high-frequency electromagnetic radiation: frequency and power dependence.

Gapeyev AB<>, Mikhailik EN<>, Chemeris NK<>.

Institute of Cell Biophysics of Russian Academy of Sciences, Pushchino, Moscow Region, Russia. gapeyev@icb.psn.ru

Abstract

Using a model of acute zymosan-induced footpad edema in NMRI mice, the frequency and power dependence of anti-inflammatory effect of low-intensity extremely high-frequency electromagnetic radiation (EHF EMR) was found. Single whole-body exposure of animals to EHF EMR at the intensity of 0.1 mW/cm(2) for 20 min at 1 h after zymosan injection reduced both the footpad edema and local hyperthermia on average by 20% at the frequencies of 42.2, 51.8, and 65 GHz. Some other frequencies from the frequency range of 37.5-70 GHz were less effective or not effective at all. At fixed frequency of 42.2 GHz and intensity of 0.1 mW/cm(2), the effect had bell-shaped dependence on exposure duration with a maximum at 20-40 min. Reduction of intensity to 0.01 mW/cm(2) resulted in a change of the effect dependence on exposure duration to a linear one. Combined action of cyclooxygenase inhibitor sodium diclofenac and EHF EMR exposure caused a partial additive effect of decrease in footpad edema. Combined action of antihistamine clemastine and EHF EMR exposure caused a dose-dependent abolishment of the anti-inflammatory effect of EHF EMR. The results obtained suggest that arachidonic acid metabolites and histamine are involved in realization of anti-inflammatory effects of low-intensity EHF EMR.

J Photochem Photobiol B.  2007 Nov 12;89(1):50-5. Epub 2007 Sep 6.

Anti-inflammatory effects of low-level laser therapy (LLLT) with two different red wavelengths (660 nm and 684 nm) in carrageenan-induced rat paw edema.

Albertini R, Villaverde AB, Aimbire F, Salgado MA, Bjordal JM, Alves LP, Munin E, Costa MS.

Source

Instituto de Pesquisa & Desenvolvimento, IP&D, Universidade do Vale do Paraíba, UNIVAP, Av. Shishima Hifumi, 2911, CEP: 12244-000 São José dos Campos, SP, Brazil.

Abstract

It has been suggested that low-level laser therapy (LLLT) can modulate inflammatory processes. The aim of this experiment was to investigate what effects red laser irradiation with two different wavelengths (660 nm and 684 nm) on carrageenan-induced rat paw edema and histology. Thirty two male Wistar rats were randomly divided into four groups. One group received a sterile saline injection, while inflammation was induced by a sub-plantar injection of carrageenan (1 mg/paw) in the three other groups. After 1 h, LLLT was administered to the paw in two of the carrageenan-injected groups. Continuous wave 660 nm and 684 nm red lasers respectively with mean optical outputs of 30 mW and doses of 7.5 J/cm(2) were used. The 660 nm and 684 nm laser groups developed significantly (p<0.01) less edema (0.58 ml [SE+/-0.17] ml and 0.76 ml [SE+/-0.10] respectively) than the control group (1.67 ml [SE+/-0.19]) at 4h after injections. Similarly, both laser groups showed a significantly lower number of inflammatory cells in the muscular and conjunctive sub-plantar tissues than the control group. We conclude that both 660 nm and 684 nm red wavelengths of LLLT are effective in reducing edema formation and inflammatory cell migration when a dose of 7.5 J/cm(2) is used.

Biofizika. 2007 Sep-Oct;52(5):947-52.

Dependence of anti-inflammatory effects of high peak-power pulsed electromagnetic radiation of extremely high frequency on exposure parameters

[Article in Russian]

Gapeev AB<>, Mikha?lik EN<>, Rubanik AV<>, Cheremis NK<>.

Abstract

A pronounced anti-inflammatory effect of high peak-power pulsed electromagnetic radiation of extremely high frequency was shown for the first time in a model of zymosan-induced footpad edema in mice. Exposure to radiation of specific parameters (35, 27 GHz, peak power 20 kW, pulse widths 400-600 ns, pulse repetition frequency 5-500 Hz) decreased the exudative edema and local hyperthermia by 20% compared to the control. The kinetics and the magnitude of the anti-inflammatory effect were comparable with those induced by sodium diclofenac at a dose of 3 mg/kg. It was found that the anti-inflammatory effect linearly increased with increasing pulse width at a fixed pulse repetition frequency and had threshold dependence on the average incident power density of the radiation at a fixed pulse width. When animals were whole-body exposed in the far-field zone of radiator, the optimal exposure duration was 20 min. Increasing the average incident power density upon local exposure of the inflamed paw accelerated both the development of the anti-inflammatory effect and the reactivation time. The results obtained will undoubtedly be of great importance in the hygienic standardization of pulsed electromagnetic radiation and in further studies of the mechanisms of its biological action.

Photomed Laser Surg.  2007 Aug;25(4):245-9.

Low-level laser therapy (GaAs lambda = 904 nm) reduces inflammatory cell migration in mice with lipopolysaccharide-induced peritonitis.

Correa F, Lopes Martins RA, Correa JC, Iversen VV, Joenson J, Bjordal JM.

Source

University Nove Julho, São Paulo, Brazil.

Abstract

OBJECTIVE:

This study was designed to study the effect of an infrared low-level laser (GaAs lambda = 904 nm, 4 mW) on inflammatory cell migration in lipopolysaccharide (LPS)-induced peritonitis in mice.

BACKGROUND DATA:

It has been suggested that red wavelengths of low-level laser therapy (LLLT) can exert anti-inflammatory effects, but little is known about the anti-inflammatory effects of infrared lasers. Peritonitis is a potentially life-threatening inflammatory condition that may be suitable for studying anti-inflammatory effects of infrared lasers.

METHODS:

Sixty male mice were randomly divided into five groups, and one group was given an intraperitoneal sterile saline injection. In the remaining four groups, peritonitis was induced by an intraperitoneal LPS injection. Animals in three of the LPS groups were irradiated at a single point over the peritoneum with doses of 3 J/cm(2), 7.5 J/cm(2), and 15 J/cm(2), respectively. The fourth group injected with LPS was an LPS-control group.

RESULTS:

At 6 hours after injection the groups irradiated with doses of 3 J/cm(2) and 7.5 J/cm(2) had a reduced number of neutrophil cells in the peritoneal cavity compared with the LPS-control group, and there were significant differences between the number of neutrophils in the peritoneal cavity between the LPS-control group and groups irradiated with doses of 3 J/cm(2) (-42%) and 7.5 J/cm(2) (-70%). In the group irradiated with 15 J/cm(2), neutrophil cell counts were lower than, but not significantly different from, LPS controls (-38%; p = 0.07). At 24 hours after injection, both neutrophil and total leukocyte cell counts were lower in all the irradiated groups than in the LPS controls. The 3-J/cm(2) exposure group showed the best results at 24 hours, with reductions of 77% in neutrophil and 49% in leukocyte counts.

CONCLUSION:

Low-level laser therapy (904 nm) can reduce inflammatory cell migration in mice with LPS-induced peritonitis in a dose-dependent manner.

Photomed Laser Surg. 2007 Apr;25(2):112-7.

Effect of low-power laser therapy on hemorrhagic lesions induced by immune complex in rat lungs.

Aimbire F<>, Lopes-Martins RA<>, Albertini R<>, Pacheco MT<>, Castro-Faria-Neto HC<>, Martins PS<>, Bjordal JM<>.

Laboratory of Animal Experimentation, Research and Development Institute (IP&D), Vale do Paraíba University (UNIVAP), São José dos Campos, Brazil.

OBJECTIVE: The aim of this study was to investigate if low-level laser therapy (LLLT) can modulate formation of hemorrhagic lesions induced by immune complex.

BACKGROUND DATA: There is a lack of information on LLLT effects in hemorrhagic injuries of high perfusion organs, and the relative efficacy of LLLT compared to anti-inflammatory drugs.

METHODS: A controlled animal study was undertaken with 49 male Wistar rats randomly divided into seven groups. Bovine serum albumin (BSA) i.v. was injected through the trachea to induce an immune complex lung injury. The study compared the effect of irradiation by a 650-nm Ga-Al-As laser with LLLT doses of 2.6 Joules/cm(2) to celecoxib, dexamethasone, and control groups for hemorrhagic index (HI) and myeloperoxide activity (MPO) at 24 h after injury.

RESULTS: The HI for the control group was 4.0 (95% CI, 3.7-4.3). Celecoxib, LLLT, and dexamethasone all induced significantly (p < 0.01) lower HI than control animals at 2.5 (95% CI, 1.9-3.1), 1.8 (95% CI, 1.2-2.4), and 1.5 (95% CI, 0.9-2.1), respectively, for all comparisons to control. Dexamethasone, but not celecoxib, induced a slightly, but significantly lower HI than LLLT (p = 0.04). MPO activity was significantly decreased in groups receiving celecoxib at 0.87 (95% CI, 0.63-1.11), dexamethasone at 0.50 (95% CI, 0.24-0.76), and LLLT at 0.7 (95% CI, 0.44-0.96) when compared to the control group, at 1.6 (95% CI, 1.34-1.96; p < 0.01), but there were no significant differences between any of the active treatments.

CONCLUSION: LLLT at a dose of 2.6 Joules/cm(2) induces a reduction of HI levels and MPO activity in hemorrhagic injury that is not significantly different from celecoxib. Dexamethasone is slightly more effective than LLLT in reducing HI, but not MPO activity.

Biofizika. 2006 Nov-Dec;51(6):1055-68.

Pharmacological analysis of anti-inflammatory effects of low-intensity extremely high-frequency electromagnetic radiation

[Article in Russian]

Gapeev AB<>, Lushnikov KV<>, Shumilina IuV<>, Chemeris NK<>.

Abstract

The anti-inflammatory effect of low-intensity extremely high-frequency electromagnetic radiation (EHF EMR, 42.0 GHz, 0.1 mW/cm2) was compared with the action of the known anti-inflammatory drug sodium diclofenac and the antihistamine clemastine on acute inflammatory reaction in NMRI mice. The local inflammatory reaction was induced by intraplantar injection of zymosan into the left hind paw. Sodium diclofenac in doses of 2, 3, 5, 10, and 20 mg/kg or clemastine in doses of 0.02, 0.1, 0.2, 0.4, and 0.6 mg/kg were injected intraperitoneally 30 min after the initiation of inflammation. The animals were whole-body exposed to EHF EMR for 20 min at 1 h after the initiation of inflammation. The inflammatory reaction was assessed over 3 – 8 h after the initiation by measuring the footpad edema and hyperthermia of the inflamed paw. Sodium diclofenac in doses of 5 – 20 mg/kg reduced the exudative edema on the average by 26% as compared to the control. Hyperthermia of the inflamed paw decreased to 60% as the dose of was increased diclofenac up to 20 mg/kg. EHF EMR reduced both the footpad edema and hyperthermia by about 20%, which was comparable with the effect of a single therapeutic dose of diclofenac (3 – 5 mg/kg). The combined action of diclofenac and the exposure to the EHF EMR caused a partial additive effect. Clemastine in doses of 0.02-0.4 mg/kg it did not cause any significant effects on the exudative edema, but in a dose of 0.6 mg/kg it reduced edema by 14 – 22% by 5 – 8 h after zymosan injection. Clemastine caused a dose-dependent increase in hyperthermia of inflamed paw at doses of 0.02-0.2 mg/kg and did not affect the hyperthermia at doses of 0.4 and 0.6 mg/kg. The combined action of clemastine and EHF EMR exposure caused a dose-dependent abolishment of the anti-inflammatory effect of EHF EMR. The results obtained suggest that both arachidonic acid metabolites and histamine are involved in the realization of anti-inflammatory effects of low-intensity


Braz Dent J (2001) 12(3): 187-190 ISSN 0103-6440

Assessment of Anti-Inflammatory Effect of 830nm Laser Light Using C-Reactive Protein Levels

André Carlos de FREITAS1
Antonio Luiz Barbosa PINHEIRO2
Paulo MIRANDA3
Fábio Albuquerque THIERS4
Alessandro Leonardo de Barros VIEIRA5

1Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, PUC-RS, Porto Alegre, RS, Brazil
2Laser Center, School of Dentistry, Federal University of Bahia, Salvador, BA, Brazil
3Laboratory of Immunopathology Keizo Asami (LIKA), Federal University of Pernambuco, Recife, PE, Brazil
4Harvard Medical School, Boston, MA, USA
5Private Dental Practice, Recife, PE, Brazil


Correspondence: Prof. Dr. Antonio Liuz Barbosa Pinheiro, Departamento de Diagnóstico e Terapêutica, Faculdade de Odontologia, Universidade Federal da Bahia, Av. Araújo Pinho, 62, Canela, 40110-150 Salvador, BA, Brasil. Tel/Fax: +55-71-336-9479. e-mail: albp@ufba.br<>

INTRODUCTION<> | MATERIAL AND METHODS<> | RESULTS<> | DISCUSSION<> | RESUMO<> | REFERENCES<>


The anti-inflammatory effect of non-surgical lasers has been proposed previously, however it was not scientifically proven. One method to assess levels of inflammation is the measurement of C-reactive protein (CRP), which is increased with the course of inflammation. The aim of this study was to assess the effect of 830nm laser irradiation after the removal of impacted third molars using the CRP as the marker of inflammation. Twelve patients were irradiated with 4.8 J of laser light per session 24 and 48 h after surgery. A control group (N=12) was treated with a sham laser. Blood samples were taken prior to, and 48 and 72 h after surgery. CRP values were more symmetric and better distributed for the irradiated group (0.320 mg/dl) than for the control (0.862 mg/dl) 48 h after surgery, however there was no statistically significant difference. After 72 h, both groups had statistically similar CRP levels (0.272 and 0.608 mg/dl), because of the normal tendency of decreasing CRP levels.

Key Words: dental surgery, inflammation, non-surgical lasers.


INTRODUCTION

Lasers have been used in several medical specialities and recently the dental profession has used laser both as a surgical tool and a biomodulating agent (1). Laser development began in 1917, when Albert Einstein proposed the principles of the stimulated emission of radiation. In 1958, Shawlow and Townes suggested the basic principles of light amplification (2).

Laser is a non-ionizing electromagnetic highly concentrated light, which, in contact with different tissues, results in several effects in tissue depending upon the wavelength and the optical properties of the irradiated tissue. Because it is a non-ionizing form of radiation, laser light can be used repeatedly within the parameters used currently because it does not induce mutagenic response. The use of laser light is not contraindicated for pregnant women, pacemaker users, and does not interfere with monitoring in the operating room (3).

Medical lasers can be classified into three main groups: Low Intensity Laser Therapy (LILT), High Intensity Laser Therapy (HILT) and Selective Laser Therapy (SLAT). LILT is also known as Low Level Laser therapy or LLLT and can modify cell metabolism, improve wound healing, reduce pain and edema, and speed inflammation and wound healing (4,5).

Inflammation occurs when immunocompetent cells are activated in response to foreign bodies or antigenic proteins. This response may have a beneficial effect (e.g. phagocytosis) or a deleterious effect (e.g. destruction of bone and cartilage in arthritis) (6).

Anti-inflammatory agents are drugs used for the treatment of non-specific inflammatory process to control signs and symptoms such as: heat, redness, pain and lack of function. There are two kinds of anti-inflammatory agents available on the market: steroids and non-steroids. The use of these drugs may result in undesirable side effects; thus, the constant search for alternative methods to control inflammation without deleterious effects to the patient.

LLLT has been used as an important tool for the control of the inflammatory process. Its anti-inflammatory effect has been studied and its ability to induce analgesia under different conditions has also been reported  (7).

The serial determination of the levels of some serum proteins may be useful for the differentiation of inflammatory and non-inflammatory conditions, as well as between clinical conditions which are known to show increased or decreased acute phase response. One of these proteins is the C-reactive protein (CRP). The magnitude of the CRP response varies directly with the severity of tissue damage, type of the inflammatory stimuli, organ or tissue involved in the process, as well as on the monitoring of the natural course of the disease and the disease response to treatment (8). CRP plasma concentration is usually low (9), increases quickly at the onset of an acute inflammatory process and quickly falls when effective control of the process occurs (10,11).


MATERIAL AND METHODS

This study was approved by the Ethical Committee of the Hospital das Clínicas  of the Universidade Federal de Pernambuco, Brazil. Twenty-four patients gave signed informed consent as determined by Brazilian regulations. The patients were selected according to the following criteria: lower wisdom tooth, older than 16 years of age and younger than 30 years of age, symptom free at the time of surgery, lack of pericoronaritis for at least 30 days prior to surgery. One week before surgery, a blood sample was taken to determine the baseline of CRP prior to surgery and irradiation. Patients with elevated CRP levels were dismissed from the study. Under local anesthesia, a maximum of two wisdom teeth were surgically removed using rotary and manual instruments. All patients received 500 mg of paracetamol after surgery. This drug was chosen because its very low anti-inflammatory action at usual doses (12). The drug was used immediately after surgery and repeated  every six hours if necessary, not exceeding 2000 mg/day.

The patients were randomly divided into two groups (even numbered patients were placed in the test group and odd numbered patients in the control group). Twenty-four and forty-eight hours after surgery, the twelve test patients received the irradiation at the Laser Center with an 830nm diode laser (40 mW; Laser Beam, Rio de Janeiro, RJ, Brazil) for a total dose of 4.8 J/cm2. Control patients were treated with a sham laser. Forty-eight and seventy-two hours after surgery, blood samples were taken and the CRP level was determined by nephelometry (13-15).

Statistical analysis was based upon the comparison between groups using the Kolmogorov-Smirnov, Mann-Whitney and Shapiro-Wilks tests (16).


RESULTS

Comparison of groups at baseline is shown in Table 1<>. Both groups had very similar levels of CRP (Kolmogorov-Smirnov = 0.17; critical value, at 5% = 0.50). Forty-eight hours after treatment (Table 2), CRP levels of irradiated patients showed a very symmetric distribution around the mean with a very small variability when compared to the controls. The Shapiro-Wilks test indicated that there was a normal distribution for the test group (S-W = 0.95; p = 0.438). However, the control group had a positive asymmetric distribution (S-W = 0.83; p = 0.021). Although there was a numerical difference between the two groups, the Mann-Whitney test did not show a significant difference between the medians of the two groups (W = 133.3; p = 0.338).

The results of the Kolmogorov-Smirnov test for two samples indicated at significance level of 5%; after 72 h the CRP levels showed the same distribution for both groups (K-S = 0.333; critical value at 5% = 0.50). Table 3 show a symmetric positive distribution for both groups. The differences of the distribution on the graphs is a reflex of the random variation.


DISCUSSION

Surgical removal of a wisdom tooth may result in edema, pain and slight bleeding. However, if these conditions are exacerbated or if there is the presence of infection or trismus, these can be considered as complications.  The lack of previous studies comparing the use of LLLT and other types of drug treatment for inflammatory conditions of the oral cavity makes it difficult to compare the results of this study. There was modification of the CRP level immediately after the surgical removal of the wisdom teeth and increased levels of CRP were found in both groups in agreement with other reports (10,11). This inflammatory response has been reported previously (17).

The level of CRP and the clinical stage of the disease have a direct relationship to the evolution of the disease and the plasma level of CRP (18). Despite the fact that several authors consider an increase of CRP levels to be an unspecific response to infection, inflammation or tissue damage (19), in this study, CRP levels were used to assess the anti-inflammatory action of the 830nm diode laser, based on other studies that used CRP levels for monitoring inflammation (17). CRP response allows monitoring of the anti-inflammatory effect of drugs. The use of aspirin, steroids, penicillin and other non-hormonal drugs will often result in a reduction of the CRP level as these drugs will suppress the underlying inflammatory process (20). However, paracetamol was used as the analgesic drug in this study because of its very low anti-inflammatory effect in usual doses (12).

The aim of post-operative therapeutics is not the suppression of inflammation, because this process is extremely important for wound healing. Therapeutic methods and drugs used in the post-operative process aim to minimize symptoms and provide more comfort for the patient. Thus, the use of a non-invasive treatment such as LLLT is a major step in clinical therapeutics because of the lack of side effects of LLLT.

Statistical analysis of the results detected a very symmetric distribution of CRP levels at the post-operative period of patients who had wisdom teeth surgically removed and were irradiated with 830nm laser light when compared to the levels observed for the controls.

Although this study failed to statistically confirm an anti-inflammatory effect of LLLT after surgical removal of wisdom teeth, the results show a reduction in the level of CRP in irradiated patients which may indicate attenuation of the inflammation; however, a larger sample is necessary to confirm this aspect. It is also important to observe that the complete treatment consisted of four minutes of irradiation within two days and that there was no additional cost for the patient, no need to use other drugs or any interference with routine life (3).


RESUMO

de Freitas AC, Pinheiro ALB, Miranda P, Thiers FA, Vieira ALB. Avaliação do efeito anti-inflamatório do laser diodo infravermelho de 830 nm através da monitorização da proteína c-reativa. Braz Dent J 2001;12(3):187-190.

A atividade anti-inflamatória da radiação Laser ainda não está muito bem estabelecida. Dentre as diferentes formas de avaliação da resposta inflamatória aguda encontra-se, a dosagem da proteína C-reativa (PCR), que na inflamação encontra-se elevada. O objetivo deste trabalho foi avaliar a capacidade anti-inflamatória do Laser Diodo Infra-Vermelho de 830nm no pós-operatório de cirurgias para remoção de terceiros molares retidos, através da análise cinética da variação dos níveis da PCR. A irradiação foi realizada a uma distância focal de 0,5cm, por um tempo total de dois minutos em corrente contínua, nos pontos preestabelecidos. As cirurgias foram realizadas no Bloco Cirúrgico do Departamento de Prótese e Cirurgia Buco Facial da Universidade Federal de Pernambuco. A população alvo foi de 24 pacientes divididos aleatoriamente em dois grupos. O grupo teste foi submetido à Laserterapia nas 24 e 48 horas pós-operatório (PO). O grupo controle foi submetido a uma falsa irradiação, com os mesmos intervalos do grupo teste. As amostras de sangue, para a dosagem da PCR foram colhidas antes da cirurgia e após 48 e 72 horas. Os valores da PCR no grupo teste 48 horas PO apresentou uma distribuição bastante simétrica em torno de sua média e com a variabilidade bem menor comparada com o grupo controle, porém não encontramos diferenças estatisticamente significante. Nas 72 horas PO os valores da PCR foram bastante semelhantes o que pode ser explicado, por uma tendência natural da queda desta proteína nesta fase.

Unitermos: cirurgia odontológica, inflamação, laser não cirúrgico.


REFERENCES

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2. Niccoli Filho WD, Okamoto T, Cardenuto N. Efeitos da radiação Laser do tipo Hélio-Neónio verde (550 nm) no processo de reparo em feridas de extração dental: estudo histológico em ratos. Rev Odontol UNESP 1993;22:213-221.
3. Pinheiro ALB.  Evolução histórica. In: Lasers na Odontologia Moderna. Brugnera Jr A, Pinheiro ALB. eds. São Paulo: Pancast; 1998. p 17-23.
4. Roynesdal AK, Bjõrnland T, Barkvoll P, Haanaes HE. The effect of soft laser application on postoperative pain and swelling. Int J Oral Maxillofac Surg 1993;22:242-245.
5. Conlan MJ, Raplay JW, Cobb CM. Biostimulation of wound healing by low-energy laser irradiation. J Clin Periodontol 1996;23:492-496.
6. Payan DG. Antiinflamatórios não-esteroides; Analgésicos não-opióides; drogas utilizadas na gota. In: Farmacologia Básica e Clínica. Katzung BG. ed. 5th edn. Rio Janeiro: Guanabara Koogan; 1994. p 368-381.
7. Genovesse WJ, Brugnera Jr A, Villa RG. Laser na odontologia. In: Metodologia do Exame Clínico em Odontologia. Genovesse WJ. ed. 2nd edn. São Paulo: Pancast; 1992. p 325-251.
8. Ohzato H, Yoshizaki M, Nishimoto N, Ogata A, Tagoh H, Ronden H, Goto MH, Kishimoto T, Mori T. Interleukin-6 as a new indicator of inflammatory status: detection of serum levels of interleukin-6 and C-reactive protein after surgery. Surgery 1992;111:201-209.
9. Crockson RA, Payne CJ, Ratcliff AP, Soothill JF. Time sequence of acute phase reactive proteins following surgical trauma. Clin Chim Acta 1966;14:435-441.
10. Hashish I, Harvey W, Harris M. Anti-inflammatory effects of ultrasound therapy: evidence for a major placebo effect. Brit J Rheumat 1986;25:77-81.
11. Shih LY, Wu JJ, Yang DJ. Erythrocyte sedimentation rate and C-reactive protein values in patients with total hip arthroplasty. Clin Orthop Relat Res 1987;225:238-246.
12. Insel PA. Fármacos, analgésicos-antipiréticos e antiinflamatórios e medicamentos usados no tratamento da gota. In: Goodman & Gilman’s As Bases Farmacológicas da Terapêutica. 9th edn. Rio Janeiro: Guanabara Koogan; 1996. p 450-480.
13. Sanchez MCA. Testes sorológicos. In: Diagnóstico Laboratorial das Principais Doenças Infecçiosas e Auto-imunes. Ferreira AW, Ávila SLM. eds. 5th edn. Rio Janeiro: Guanabara Koogan; 1996. p 7-28.
14. Davis ML, Austin C, Messmer BL, Nichols WK, Bonin AP, Bennett MJ. IFCC – Standardized pediatric reference intervals for 10 serum proteins using the Beckman Array 360 System. Clin Biochem 1996;29:489-492.
15. Check IJ, Papadea C.  Imunoglobulin  quantitation. In: Manual of Clinical Laboratory Immunology. Rose NR, Macario EC, Folds JD, Clifford Lane H, Nakamura RM. eds. 5th ed. Washington: American Society for Microbiology; 1997. p 136-138.
16. Conover WJ. Pratical Nonparametric Statistics. 2nd edn. New York: John Wiley & Sons; 1980. p 493.
17. Palosuo T, Husman T, Koistinen J, Aho K. C-reactive protein in population samples. Acta Med Scand 1986;220:175-179.
18. Harmoinen A, Hällström O, Grönroos P. Rapid quantitative determination of C-reactive protein using laser-nephelometer. Scand J Clin Invest 1980;40:293-295.
19. Grützmeier S, Schenck H. Four immunochemical methods for measuring C-reactive protein in plasma compared. Clin Chem 1989;35:461-463.
20. Chwals WJ, Fernandez ME, Jamie AC, Charles BJ. Relationship of metabolic indexes to postoperative mortality in surgical infants. J Ped Surg 1993;28:819-822.

Accepted March 13, 2000