Long-Term Safety of Low-Level Laser Therapy at Different Power Densities and Single or Multiple Applications to the Bone Marrow in Mice

Abstract

Objective: The purpose of this study was to determine the long-term safety effect of low-level laser therapy (LLLT) to the bone marrow (BM) in mice. Background data: LLLT has been shown to have a photobiostimulatory effect on various cellular processes and on stem cells. It was recently shown that applying LLLT to BM in rats post-myocardial infarction caused a marked reduction of scar tissue formation in the heart. Methods: Eighty-three mice were divided into five groups: control sham-treated and laser-treated at measured density of either 4, 10, 18, or 40 mW/cm² at the BM level. The laser was applied to the exposed flat medial part of the tibia 8 mm from the knee joint for 100 sec. Mice were monitored for 8 months and then killed, and histopathology was performed on various organs. Results: No histological differences were observed in the liver, kidneys, brain or BM of the laser-treated mice as compared with the sham-treated, control mice. Moreover, no neoplasmic response in the tissues was observed in the laser-treated groups as compared with the control, sham-treated mice. There were no significant histopathological differences among the same organs under different laser treatment regimes in response to the BM-derived mesenchymal stem cell proliferation following LLLT to the BM. Conclusions: LLLT applied multiple times either at the optimal dose (which induces photobiostimulation of stem cells in the BM), or at a higher dose (such as five times the optimal dose), does not cause histopathological changes or neoplasmic response in various organs in mice, as examined over a period of 8 months.

Introduction

L ow-level laser therapy (LLLT) has been shown to modulate various biological processes. In the past decade, LLLT has also been applied to various internal organs (heart, brain, liver) in order to modulate processes in these organs post-injury or ischemia.^{1

–8,9} In the application of LLLT to treat stroke, there has been a shift from extensive work on experimental animal models⁸ to well-controlled human clinical trials, in which the LLLT is applied transcranially to the stroked brain.^9

–12 The safety and possible toxic effects of transcranial LLLT to the rat brain have been reported previously.^13
–15 The conclusion of those studies was that laser light (808 nm) appears to be safe, with no side effects observed up to 1 year following treatment, at single or multiple power densities up to 50 mW/cm² of the laser to the surface of the cerebral cortex in the brain. This safe feature of the laser application was maintained even when a dose of up to five times the optimal dose was applied to the rat brain.^13
–15 In clinical trials, it was also evident that there was no increase in side effects in the laser-treated patients post-stroke as compared with control placebo patients.^10
–12

We recently reported that LLLT application to the bone marrow (BM) of rats post-myocardial infarction (MI) caused a marked reduction in scarring, possibly because of the induction of stem cells in the BM and their migration to the infarcted area.^16,17 The BM hosts a variety of cell types including various progenitor cells of the mesenchymal and hematopoietic system. The possible long-term toxic effects of LLLT application to the BM have not been studied to date, and such study is therefore warranted in light of possible clinical application of the new approach to the induction of stem cells by LLLT.

The present study was designed to investigate the long-term safety of laser application to the BM on the histopathology of the BM, liver, kidneys, and brain of mice that underwent LLLT at several different doses and frequencies, compared with control, sham-treated groups of mice.

Materials and Methods

Experimental procedures

A total of 83 Imprinting Control Region (ICR) mice, weighing 20 g (6 weeks old), were used in this experiment. All the experimental procedures were approved by the Animal Care Committee of Tel-Aviv University. The mice were divided randomly into five groups. Bone marrow was sham treated or laser treated at various power densities as detailed (see “Laser application” section). All mice were kept under optimal conditions (food and water provided ad libitum) at room temperature (22–24°C). Mice were killed 8 months post-laser treatment and mortality was recorded daily to calculate the survival rate over 8 months.

Laser application

In control sham-treated mice (n=35) the laser was placed on the tibia but not turned on. Four groups of mice were laser treated at various power densities at the level of the BM as follows: 4 mW/cm² (n=8), 10 mW/cm² (n=16), 18 mW/cm² (n=6), and 40 mW/cm² (n=18). All laser treatments were given for a duration of 100 sec. Laser treatment was applied twice weekly for the first 2 weeks at the abovementioned doses, up to a period of 8 month.

Mice were randomly assigned to a laser-treated or control sham-treated group. A diode (Ga-Al-As) laser, wavelength 804 nm with a tunable power output of maximum of 400 mW (Lasotronic Inc., Zug, Switzerland) served for application of LLLT to the BM, as described by us previously.¹⁶ A metal backed glass fiberoptic (30 mm long and 2 mm in diameter) was connected to the power output of the laser device. The distal flat end of this fiberoptic was placed directly on the bony proximal medial part of the tibia bone following a small incision (5 mm) through skin and muscle. The measurements of power density and transmission of the laser through the tibia bone were performed as detailed (see Fig. 1). In a preliminary experiment, three intact mice (same age and weight as the experimental laser-treated mice) were killed by overdose of avertin; the fresh tibia bone was removed, cleaned from attached muscles and tendons, and then carefully cut on its longitudinal axis to yield two sections of bone featuring the medial and lateral parts of the tibia. The medial flat aspect of the bone was cleaned from the BM and used for measurement of laser transmission through the bone. The distal tip of the laser fiberoptic was placed perpendicular to the flat aspect of the tibia, ∼3 mm distal to the in the knee joint. The tunable laser was set to provide various power outputs on its distal tip (see Table 1 for details). The laser meter probe (model PD.300-3W, Ophir Inc. Jerusalem, Israel) was then placed on the opposite side of the bone (inside the bone where the BM is normally located), and the power was measured (Fig. 1). The irradiation was performed using multimode fiberoptics 1.5 mm in diameter. The laser beam profile was measured using the truncated Gaussian beam method according to Urey¹⁸ at the 1/e² location on freshly prepared tibia as described. The fiber output Gaussian beam had a numerical aperture of 0.22, that is, a divergence of ∼±13^o from its central axis. Measurement of the energy at the output of the fiber appears in Table 1, and the spot size, measured at 1/e² of the energy curve, starts at 1.5 mm diameter (see Fig. 1 for further clarification). The diameter of the beam at the BM interface with the bone is ∼1 mm away from the fiber distal end giving a calculated 1/e² beam diameter of 1.94 mm, and actually is even larger because of the scattering of light in the bone. The laser transmission through the bone was calculated as the ratio value (in percent) of the power output measured after laser penetration through the bone divided by the power at the tip of the fiberoptic on the external surface of the tibia (see Fig. 1 and Tables 1 and 2 for details).

FIG. 1.

Schematic presentation of (a) laser beam dispersion at bone and bone marrow, (b) measurements of power output at the distal end of the fiberoptic with the calorimeter, and (c) measurement and dispersion of the laser beam after the bone and at the bone level.

Table 1.

Summary of Parameters at the Bone Marrow

Wavelength (nm)	804	804	804	804
Output (mW)	11.2	2.8	5	1.12
Illuminated area (cm²)	0.28	0.28	0.28	0.28
Irradiance (mW/cm²)	4	10	18	40
Time (seconds)	100	100	100	100
Energy (Joules)	0.112	0.28	0.5	1.12
Fluence (J/cm²)	0.4	1	1.8	4

Table 2.

Summary of Parameters at the Bone Surface

Wavelength (nm)	804	804	804	804
Output (mW)^a	4.1	10.3	18.5	40
Illuminated area (cm²)^b	0.018	0.018	0.018	0.018
Irradiance (mW/cm²)	227	572	1027	2222
Time (sec)	100	100	100	100
Energy (Joules)	0.41	1.03	1.85	4
Fluence (J/cm²)	23	57	103	222

Measured at 1/e² of energy peak.

The area of the beam where the diameter is measured at 1/e² of the peak energy.

At the abovementioned setting, the laser beam diameter on the BM was 6 mm, comprising an area of 0.28 cm², the measured transmission of the laser through the tibial bone was 27%, and the tunable laser power output from the distal tip of the fiberoptic (delivered directly to the external surface of the tibia) was set to provide parameters of 4, 10, and 18 and 40 mW/cm² power densities on the BM tissue, which comprised 0.4, 1, 1.8, and 4 J/cm², respectively. Laser application was applied for 100 sec in all rats.

Histology

Mice were killed by overdose of avertin. The tibia bone, liver, kidneys, and brain were excised and washed for 30 sec in extensive saline to remove external blood. The tissue samples (∼0.5×0.5×0.5 cm of tissue randomly cut from each organ) were fixed in 10 mL of 4% neutral buffered formalin solution for 3 days. The formalin solution was replaced with a fresh one after 24 h. The fixative was washed in running tap water for 3 h, and then dehydration was performed with graded 50–100% ethanol solutions. The tissue samples were then embedded in paraffin and 8 μm sections were prepared using a microtome. Defined cross-section samples (2 mm thick) were taken from the abovementioned organs for histology. Eight micron paraffin sections were prepared from the tissue samples of each organ. Sections were stained with hematoxylin-eosin. Two observers, blinded to whether the mice were control or laser treated, reviewed the histological sections.

Isolation and culturing of mesenchymal stem cells (MSCs) from bone marrow

Bone marrow of control, sham-operated and laser-treated mice, at power densities of 4, 18, and 40 mW/cm² was extracted from the tibias and femurs of each mouse using a steel rod as described by us previously,¹⁶ and pooled for each mouse. Bone marrow was not extracted from the mice treated at a power density of 10 mW/cm². Mesenchymal stem cell isolation was performed essentially as described by Davani et al.¹⁹ The collected BM was incubated in a shaker with 5 mL Dulbecco Modified Eagle Medium (DMEM) containing type I collagenase (250 U/mL, Sigma Israel) for 45 min at 37°C. Cells were then cultured at a concentration of 1.3×10⁶ cm² in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mmol/l L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin (Biological Industries, Beit Haemek, Israel) in small tissue culture flasks. Forty-eight hours later, the medium was replaced to remove nonadherent cells (MSCs are adherent¹⁸), and thereafter replaced every 4 days. C-kit+ staining of these cultures revealed that ∼90% of the cells were positively immunostained for c-kit. The isolated MSCs from the mice were harvested for 7 days (4 days are necessary for cells to adhere, and an additional 3 days are needed to express their proliferative capacity). After 7 days in culture, the cells were harvested and centrifuged, stained with toluidine blue 0.1%, and counted using a hemocytometer.

Statistical analysis

The SigmaStat 2.0 (Sigma, St Louis, MO) software was used for statistical analysis. Tests were performed first for normality distribution, followed by parametric (Student's t test) test.

Results

No histological differences were observed in the liver, kidneys, brain, or BM of the laser-treated mice as compared with the sham-treated control mice (Fig. 2). Moreover, no neoplasmic response in the tissues was observed in the laser-treated groups as compared with the controls.

FIG. 2.

Representative light micrographs of kidney (a–c), brain (d–f ), bone marrow (BM) (g–i), and liver (j–l) cross-sections of sham-treated control (non–laser-treated) mice (a, d, g, j) and laser-treated (to BM) mice at power densities of 10 mW/cm² (b, e, h, k) and 50 mW/cm² (c, f, i, l)×200.

There was no statistical difference (Fisher's test) in mortality during the 8-month follow-up period between the control group and any of the laser-treated groups.

The mean number of MSCs that were isolated from non-laser-treated mice and grown in culture for 7 days was 9×10⁵±2, not significantly different from the mean number of the MSCs (8.4×10⁵±2, 6.6×10⁵±2, and 6.5×10⁵±2) that were isolated from the BM of laser-treated mice at power densities of 4, 18, and 40 mW/cm², respectively (Fig. 3).

FIG. 3.

Effect of low-level laser therapy (LLLT) applied to the tibia of mice, on the number of mesenchymal stem cells (MSCs) isolated from the tibia 8 weeks post-LLLT. The entire bone marrow (BM) from control non-laser-treated (open column) or laser-treated (solid column) tibias was collected and pooled together for each experimental group and then grown in six culture wells. The MSCs that were isolated from each group were grown in culture for 7 days and then counted. Results are mean±SEM of six repetitions (wells) in each group.

Discussion

The results of the present study indicate that applying LLLT at a power density dose of up to five times the optimal dose that causes a photobiostimulatory effect on the cells in the BM of rats post-MI,⁵ did not induce any pathological effects on the various studied organs in the mice. Histological examination of these organs did not reveal any neoplasmic response over a period of 8 months (almost the entire lifespan of mice) following the application of LLLT to the BM.

The results corroborate a previous long-term safety study on the application of LLLT to the rat brain, which found no toxicological abnormalities in the brain at similar laser power densities to those used in the present study.^9,12
–14 In that study, however, only the brain tissue was analyzed. In the present study, we looked for pathological changes in several organs, as the cells that populate the BM and were induced by means of LLLT might have migrated through the circulation to other organs. The present study is the first to explore possible long-term histopathological changes concomitantly in several organs following multiple LLLT applications to the BM. In another short-term study of LLLT application to the brain, at power densities similar to those in the present study, no side effects were observed. Moreover, when LLLT was applied to the brain post-traumatic brain injury, no changes in the histopathology of the brain were evident.^20,21

Another indication of the safety of LLLT to the BM was found previously when the kinetics of growth of MSCs isolated from laser-treated or non-laser-treated BM were followed in vitro.¹⁶ It was found that MSCs isolated from the BM of infarcted rats 4 weeks post-LLLT had a significant twofold higher rate of proliferation than the non-laser-treated BM. However, this enhanced proliferation was not observed when the MSCs were isolated from the BM 6 weeks post-LLLT. These results indicate that the induction of cell proliferation by LLLT is limited to a relatively short period post-laser application, and that there is no discernible neoplasmic response in the stem cells in the BM that have been induced to proliferate at a faster rate by the LLLT.

In an additional study, we followed the kinetics of the growth factors inducible nitric oxide (iNOS) and vascular endothelial growth factor (VEGF) in rats with post-MI where the laser was applied directly to the heart.²² The content of these factors was higher in the laser-treated hearts 2–4 days post-MI than in the non-laser-treated hearts, but was identical to the non-laser-treated hearts at 7 days post-MI. The above finding indicates that LLLT does not induce production of these significant growth factors in an uncontrolled manner that differs from their production post-MI without the interference of LLLT.

In the present study, we have also shown that the number of MSCs isolated from the BM of laser-treated and non-laser-treated mice (at 8 months of age and before euthanasia) proliferated in culture to a similar extent. This gives credence to a non-neoplasmic long-term possible effect of LLLT on the BM. This phenomenon, in addition to the histopathology of the BM, which did not indicate neoplasmic response in the laser-treated mice, supports the long-term safety of LLLT application to the BM in mice.

Conclusions

It is concluded that LLLT applied multiple times either at the optimal power and energy density (that induces photobiostimulation of stem cells in the BM), or at higher power densities up to five times the optimal (that yields photobiostimulatory effects), does not cause histopathological changes or neoplasmic response in the various organs of the mice as examined over a period of 8 months.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

References

Streeter

, De Taboada

, Oron

2004. Mechanisms of action of light therapy for stroke and acute myocardial infarction. Mitochondrion, 4:569–576.

Oron

2006. Photoengineering of tissue repair in skeletal and cardiac muscles – Review. Photomed. Laser Surg., 24:111–120.

Tuby

, Maltz

, Oron

2011. Induction of autologous mesenchymal stem cells in the bone marrow by low-level laser therapy has profound beneficial effects on the infarcted rat heart. Laser. Surg. Med., 43:401–409.

Karu

2007. Ten lectures on basic science of laser phototherapy. Gragesberg, Sweden: Prima Books.

Karu

2010. Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP. Photomed. Laser Surg., 28:159–160.

Oron

, Yaakobi

, Oron

et al. 2001. Low energy laser irradiation reduces formation of scar tissue following myocardial infarction in dogs. Circulation, 103:296–301.

Oron

, Maltz

, Tuby

, Sorin

, Czerniak

2010. Enhanced liver regeneration following acute hepatectomy by low level laser therapy. Photomed. Laser Surg., 28:675–678.

Oron

, Oron

, Chen

et al. 2006. Low level laser therapy applied transcranially to rats following induction of stroke significantly reduces long-term neurological deficits. Stroke, 37:2620–2624.

DeTaboada

, Ilic

, Leichliter

, Oron

, Streeter

2006. Transcranial application of low energy laser Irradiation improves neurological deficits in rats following acute stroke. Lasers Surg. Med., 38:70–73.

10.

Lampl

, Zivin

J.A.

, Fisher

et al. 2007. Infrared laser therapy for ischemic stroke: a new treatment strategy: results of the neurotheral effectiveness and safety trial-1 (NEST-1) Stroke, 38:1843–1849.

11.

Zivin

J.A.

, Albers

G.W.

, Bornstein

et al. 2009. Effectiveness and safety of transcranial laser therapy for acute ischemic stroke. Stroke, 40:1359–1364.

12.

Zivin

J.A.

, Albers

G.W.

, Bornstein

et al. 2009. Neuro Thera Effectiveness and Safety Trial-2 Investigators. Effectiveness and safety of transcranial laser therapy for acute ischemic stroke. Stroke, 40:1359–1364.

13.

Ilic

, Leichliter

, Streeter

, Oron

, DeTaboada

, Oron

2006. Effects of power densities, continuous and pulse frequencies, and number of sessions of low-level laser therapy on intact rat brain. Photomed. Laser Surg., 24:458–466.

14.

Lapchak

P.A.

, Han

M.K.

, Salgado

K.F.

, Streeter

, Zivin

J.A.

2008. Safety profile of transcranial near-infrared laser therapy administered in combination with thrombolytic therapy to embolized rabbits. Stroke, 39:3073–3078.

15.

McCarthy

, De Taboada

, Hildebrandt

P.K.

, Ziemer

E.L.

, Richieri

S.P.

, Streeter

2010. Long-term safety of single and multiple infrared transcranial laser treatments in Sprague–Dawley rats. Photomed. Laser Surg., 28:663–667.

16.

Tuby

, Maltz

, Oron

2011. Induction of autologous mesenchymal stem cells in the bone marrow by low-level laser therapy has profound beneficial effects on the infarcted rat heart. Lasers Surg. Med., 43:401–409.

17.

Oron

2011. Light therapy and stem cells: a therapeutic intervention of the future. Interv. Cardiol., 3:627–629.

18.

Urey

. 2004. Spot size, depth-of-focus, and diffraction ring intensity formulas for truncated Gaussian beam. Appl. Opt., 43:620–625.

19.

Davani

, Marandin

, Mersin

et al. 2003. Mesemchymal progenitor cells differentiate into an endothelial phenotype enhance vascular density and improve heart function in rat cellular cardiomyoplasty model. Circulation, 108:II-253–II-258.

20.

Ilic

, Leichliter

, Streeter

, Oron

, DeTaboada

, Oron

2006. Effects of power densities, continuous and pulse frequencies and number of sessions of low level laser therapy on intact rat brain. Photomed. Laser Surg., 24:458–466.

21.

Oron

, Oron

, Chen

et al. 2006. Low level laser therapy applied transcranially to rats following induction of stroke significantly reduces long-term neurological deficits. Stroke, 37:2620–2624.

22.

Tuby

, Maltz

, Oron

2006. Modulations of VEGF and iNOS in the rat heart by low level laser therapy are associated with cardioprotection and enhanced angiogenesis. Lasers Surg. Med., 38:682–688.