Low-Level Laser Therapy to the Bone Marrow Ameliorates Neurodegenerative Disease Progression in a Mouse Model of Alzheimer's Disease: A Minireview

Abstract

Objective: This communication reviews the ability of low-level laser therapy (LLLT) to stimulate mesenchymal stem cells (MSCs) in autologous bone marrow (BM) to enhance the capacity of MSCs to infiltrate the brain, clear β-amyloid, and improve cognition. Background: We recently reported that LLLT applied to the BM enhanced the proliferation of MSCs and their mobilization toward the ischemic heart region, suggesting a possible application of this approach in regenerative medicine and neurodegenerative diseases. It was also shown that circulating monocytes can infiltrate the brain and reduce brain amyloid load in an Alzheimer's disease (AD) mouse model. Methods and Results: MSCs from wild-type mice stimulated with LLLT demonstrated an increased ability to maturate toward a monocyte lineage and to increase phagocytosis of soluble Aβ in vitro. Furthermore, weekly LLLT for 2 months to the BM, starting at 4 months of age (progressive stage of the disease in these 5XFAD transgenic male mice), improved memory and spatial learning, compared to a sham-treated AD mouse model. Histology revealed a significant reduction in Aβ brain burden in the laser-treated mice compared to the nonlaser-treated ones. Conclusions: The application of LLLT to the BM is suggested as a therapeutic approach in progressive stages of AD, and its potential role in mediating MSC therapy in brain amyloidogenic disease is implied.

Alzheimer's disease (AD) affects more than 18 million people worldwide and is characterized by progressive memory deficits, cognitive impairment, and personality changes. The main cause of AD is generally attributed to the increased production and accumulation of amyloid-beta (Aβ), in association with neurofibrillary tangle formation that leads to neuronal death. This process mainly affects the hippocampus, which is critical for learning and memory.¹ The presence of stem cells in this structure has led to an increased interest in the phenomenon of adult neurogenesis and its role in hippocampal functioning.² Many known factors impacting neurogenesis in the hippocampus are implicated in the pathogenesis of AD.³ Since neurogenesis is modifiable, stimulation of this process, or the potential use of stem cells, recruited endogenously or implanted by transplantation, has been speculated as a possible treatment for neurodegenerative disorders, such as AD.

It was previously demonstrated that the source of new brain cells might be local, from either the subventricular zone of the forebrain⁴ or the subgranular zone of the hippocampus.⁵ The possibility that stem cells can be derived from a peripheral zone of the brain, such as from the bone marrow (BM), was also explored.⁶ Similarly, it was recently shown that human periapical cysts occurring in odontogenic cystic lesions contain mesenchymal stem cells (MSCs) with a capacity for self-renewal and multilineage differentiation.⁷ In the same study, it was shown that following exposure of these cells to neurogenic differentiation conditions, their neurogenic phenotype was expressed indicating that these cells might constitute another source of neural glial cells for cell-based therapies. In another study, it was demonstrated that dental pulp stem cells in the presence of embryonic midbrain cues display an efficient propensity toward becoming functional dopaminergic cell type.⁸

The BM features several different types of pluripotent cells: hematopoietic stem cells, MSCs, endothelial progenitor cells, side population cells, and multipotent adult progenitor cells. Like other stem cells, MSCs are capable of multilineage differentiation from a single cell and in vivo functional reconstitution of injured tissues.⁹ One of the properties of stem cells is their capacity to migrate to one or more appropriate microenvironments.¹⁰ Certain stem cells are able to exit their production site and circulate in the blood before reseeding in their target tissues. For MSCs, the nature of the homing sites and their kinetics in circulating peripheral blood are still under debate. However, following infusion, MSCs have been found in multiple tissues, leading to the hypothesis that they have the ability to home and adjust their differentiation pathways to diverse tissue microenvironments.¹¹ It was recently shown that intracerebral transplantation of bone marrow-derived MSCs into the brain of an induced AD model reduced the Aβ protein levels and accelerated the activation of microglia cells, compared to sham-transplanted animals. Furthermore, it was suggested that blood-derived microglia-like cells have the ability to eliminate amyloid deposits by means of a cell-specific phagocytic mechanism.¹²

Low-level laser therapy (LLLT) has been found to have a photobiomodulation (PBM) effect on various biological processes.^13,14 The PBM of processes in the brain by LLLT has been addressed by several studies. Transcranially applied LLLT has been shown to have beneficial effects in rats, rabbits, and humans poststroke.^15
–17 Furthermore, LLLT applied multiple times transcranially to AD mice results in improved neurological function in those mice over nonlaser-treated ones.¹⁸

The PBM of stem cells or progenitor cells by LLLT has not been extensively studied. Low-level laser application to adipose-derived stem cells attenuated their activity.^19,20 Laser application to normal human neural progenitor cells significantly increased ATP production in these cells.²¹ LLLT was found also to significantly increase survival and/or proliferation of MSCs postimplantation into the ischemic heart, followed by a marked reduction of scarring and enhanced angiogenesis.²² Furthermore, LLLT applied to autologous BM in the infarcted heart of rats led to a 79% reduction in the extent of scarring in the heart post-MI.^23,24 This phenomenon could be partially attributed to a higher extent of the laser-induced MSC from the BM being mobilized to the infarcted area. The rationale behind the attempt to use LLLT to induce the many cell types in the BM was that one cannot significantly affect the complex process post-MI or ischemic injury in other organs with a single type of stem cell. The native BM is known for its many types and subtypes of stem cells, which are defined by their reactivity to various antibodies. The BM also contains many progenitor cells (i.e., monocytes) that can further differentiate, for example, to macrophages. Macrophages recently have been shown to play a crucial role in mitigating the scarring process post-MI. Thus, LLLT may induce various types of cells concomitantly in the BM, which will increase in number in the blood circulation, following their enhanced proliferation in the BM. These cells will probably, eventually, and to a certain extent and under certain circumstances, home in on the ischemic zone in the ischemic organ (e.g., the heart). In a recent study,²⁵ we sought to determine whether LLLT to the BM can activate a beneficial immune response or induce stem cells to home in on the brain at progressive stages of an AD mouse model.

We first sought to evaluate the ability of laser-treated MSCs to phagocytose Aβ proteins. Isolation of MSCs was performed essentially as described previously.²² Cells were then seeded in 24-well culture plates at a concentration of 1.3 × 10⁶ cell/cm² for 1 week (medium was changed 48 h postseeding) as described previously.²⁴ The cultured MSCs were exposed to the Ga-Al-As laser for 20 sec at a power density of 50 mW/cm² to yield 1.0 J/cm² energy density. Another set of six well plates containing MSCs were sham exposed (control) to the laser (cells treated as above, but the laser was not turned on). The laser-treated and the control MSCs were left in the incubator for 3 days postlaser treatment and then incubated until 70% confluence. For phagocytosis of Aβ, MSCs were labeled with the anti-CD11b antibody as described previously,²⁶ and the percentage of Aβ phagocytosis was analyzed by fluorescence-activated cell sorting.

A significant (p = 0.041) 35% increase in phagocytosis of Aβ (1–42) was found in the cells that were laser treated, compared to the nonlaser-treated cells. Furthermore, a significant (p < 0.0001) 10% increase was detected in the CD11b activation marker of monocyte-derived cells. These results suggest that laser application to monocytes or other cell types that demonstrate phagocytotic activity, among the MSC population in the BM, can cause significant activation of these cells and, hence, enhance their capacity to uptake specifically Aβ proteins accumulated in the brain in AD mice. Indeed, it has been suggested that LLLT may affect the immune system.²⁷ Furthermore, it was shown that LLLT decreased inflammatory cytokines while upregulating nitric oxide in lipopolysaccharide-treated macrophages.²⁸

The ability of macrophages to act as phagocytes was also found to be modulated under the application of LLLT.^27,28 Thus, in our recent study,²⁵ we demonstrated a significant elevation in the activation of immune cells, detected by CD11b in MSC, following LLLT. Furthermore, we showed an increase in MSC reactivity to phagocytose soluble neurotoxic Aβ, which tends to lead to toxic oligomers in the brain. It can be hypothesized that these cells can be activated in the BM, migrate to the circulating blood, and then infiltrate to the brain and reduce the amyloid burden there. Indeed, it was previously suggested that the migration of peripherally derived mononuclear cells leads to clearance of amyloid load in an AD mouse model and improves cognition.¹² Moreover, several studies have suggested that activation of peripheral monocyte-derived macrophages, bone marrow-derived cells, and microglia can play a role in the clearance of brain amyloids in an AD mouse model.^12,29
–31

Following the findings regarding the enhanced capacity of the laser-induced CD11b-positive cells in the BM to phagocytose neurotoxic soluble Aβ in vitro, we conducted an in vivo study in an AD mice model. For this mouse model, 5XFAD transgenic male mice (Tg6799) generations were used. 5XFAD mice demonstrate amyloid burden starting from 2 months of age. By the age of 4 months, this mouse model features a high amyloid load, commencing in the cortex and expanding to the hippocampus. At 6 months of age, the mice demonstrate a robust amyloid burden in both the cortex and the hippocampus.

In this study, we sought to investigate the effect of LLLT at the progressive stage of the disease. Four-month-old AD mice were therefore treated every 10 days for 2 months with LLLT (totaling six treatments) to their BM. A tunable laser (Ga-Al-As laser, wavelength 808 nM) with power output of maximum of 400 mW was used. LLLT to the BM was performed as described previously.²² The power density of the laser beam was 10 mW/cm² on the BM, and duration of irradiation was 100 sec to yield 1.0 J/cm² energy density. The control mice underwent the same procedure as the laser-irradiated group, but the laser was not turned on. Mice were divided into three groups: AD mice treated with LLLT every 10 days; sham-operated AD mice; and intact WT mice of the same strain as the AD mice. At the age of 6 months, the mice were subjected to behavioral and cognitive tests and then sacrificed and the brain fixed and processed for β-amyloid burden in the brain. Two neurobehavioral tests were used: the object recognition test (ORT) and the contextual fear-conditioning test (FCT). ORT is distinguished by more time spent interacting with the novel object. Memory was operationally defined by the discrimination index for the novel object as the proportion of time the mice spent investigating the novel object in comparison with the familiar one.

In the FCT test, male mice were subjected to an unconditioned electric stimulus in a presession training. Twenty-four hours later, FCT was measured by scoring freezing behavior (the absence of all but respiratory movement) for 180 sec, using a freeze-frame automated scoring system. Following the neurobehavioral tests, the brains (left hemisphere) were cut into sagittal sections with a cryostat at −20°C and used for histological examination. The slices were stained with Congo-red staining and anti-Aβ and visualized by fluorescence microscopy for quantification of amyloid depositions. The hippocampal Aβ burden was presented as the percentage of insoluble total Aβ and Congo-red-positive region of the entire hippocampus region.

The results from the ORT revealed that application of LLLT to the BM of AD mice significantly elevated the percentage of time spent near the new object almost to the level of that of the WT mice. The WT 6-month-old mice demonstrated an average of 73% ± 4.11% of their time spent around a new object. This value was significantly (p < 0.01) reduced to 47.3% ± 5.58% in the group of 6-month-old and nonlaser-treated AD mice, indicating a significant memory loss in the latter. However, the average percentage of time spent near a new object in the laser-treated mice was 68.7% ± 3%, suggesting that the multiple LLLT applications to the BM of AD mice had led to recovery of their memory loss. There was no statistical difference in the time spent near a new object between the WT mice and the AD mice treated by LLLT.

The FCT nonlaser-treated AD mice showed poorer cognitive abilities (11.6 ± 4.6 sec) than the WT mice (71.1±4.6sec).The laser-treated AD mice showed a significantly increased freezing time of 40.4 ± 5.28 sec, compared to the nonlaser-treated mice. These results indicate a significantly enhanced cognitive ability and memory gain in the laser-treated BM mice, compared to the control mice. Amyloid burden in the brains of the AD mice after 2 months of treatment with the LLLT was also found to correlate with the in vitro and behavior tests. The percentage of Aβ burden, as revealed from the histology in the hippocampus region of the nonlaser-treated mice, was 180 ± 15, whereas in the laser-treated mice there was a significant reduction of 68% (p < 0.05) in Aβ burden relative to the control mice.

This study²⁵ has thus demonstrated that LLLT applied to autologous BM induces MSC activation toward phagocytosis of toxic Aβ, leading to a cognitive function improvement in an AD mouse model. It was found that LLLT treatment led to a significant reduction in brain amyloid load following a short period of treatment, starting at the late progressive disease stage. Furthermore, LLLT treatment improved cognitive behavior in the laser-treated AD mice, compared to nontreated mice at an advanced and progressive stage of AD disease. These results also correlate with the general beneficial effect of applying LLLT transcranially to AD mice.¹⁸ However, in the current study, the laser was applied for a shorter period of time and with a less frequent application than in the study by De Taboada et al.¹⁸ In addition, LLLT was applied in this study to autologous BM cells as a target organ that is remote from the impaired brain.

The amyloid burden (at 6 months) in the AD mouse model was found to be significantly reduced in the LLLT-treated mice, compared to the control mice. The results of the behavioral tests in this study are in concert with a reduction of amyloid burden in the brain. They indicate a significantly improved cognitive ability and memory in the laser-treated mice over the nonlaser-treated ones. It should be noted that regarding the ORT, the mice that received multiple applications of LLLT to the BM between 4 and 6 months of age demonstrated a significant improvement that reached the level of the cognitive ability of the WT mice.

The present study also has clinical relevance. The safety of LLLT application (at a similar power density as in the current study) has been reported in experimental animals and in double-blinded studies in humans postacute stroke.^15,32 Moreover, we recently demonstrated that LLLT application even at higher power densities to the BM of mice did not cause any histological changes in various organs over a period of almost their entire life span.³² It may thus be assumed that the application of LLLT to the BM in humans will be safe. Our ability to demonstrate that LLLT application to the BM improves cognitive brain function and reduces plaque concentration in the brain of AD mice, even when treatment is commenced at a progressive stage, is of significance. It suggests that LLLT could be applied to humans with AD, which is usually only diagnosed when already at a progressive stage.

The novel approach presented here, of the use of stem cells for cell therapy to the infarcted heart, avoids the need to isolate stem cells, to grow them in vitro, and to inject them back into the patients. It also avoids the massive loss of cells involved in cell implantation/injection due to insufficient seeding of cells or cell death shortly after implantation. The approach in the present study also overcomes the problem of growing autologous stem cell cultures, determination of the optimal amount of implanted cells, and the optimal timing for their delivery.

In conclusion, our results indicate that the novel approach of applying LLLT to autologous BM of AD mice induces the production of stem cells and immune cells, which are then recruited to the brain, demonstrating the possibility of the patient's own abilities to initiate a regenerative response in an organ, leading to a marked beneficial effect. LLLT thus offers a potential therapeutic strategy in treating symptoms of AD and perhaps also other neurodegenerative diseases.

Footnotes

Acknowledgment

The authors thank Ms. Naomi Paz for editorial assistance.

Author Disclosure Statement

No competing financial interests exist.

References

Selkoe

. Cell biology of protein misfolding: the examples of Alzheimer's and Parkinson's diseases. Nat Cell Biol, 2004; 6:1054–1061.

Morgan

. Amyloid, memory and neurogenesis. Exp Neurol, 2007; 205:330–335.

Rodriguez

, Verkhratsky

. Neurogenesis in Alzheimer's disease. J Anat, 2011; 219:78–89.

Alvarez-Buylla

, Garcia-Verdugo

, Mateo

, Merchant-Larios

. Primary neural precursors and intermitotic nuclear migration in the ventricular zone of adult canaries. J Neurosci, 1998; 18:1020–1037.

Reznikov

. Cell proliferation and cytogenesis in the mouse hippocampus. Adv Anat Embryol Cell Biol, 1991; 122:1–74.

Munoz

, Stoutenger

, Robinson

, Spees

, Prockop

. Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proc Natl Acad Sci U S A, 2005; 102:18171–18176.

Kanafi

, Majumdar

, Bohnde

, Gupta

, Datta

. Midbrain dues dictate differentiation of human dental pulp stem cells towards functional dopaminergic neurons. J Cell Physiol, 2014; 229:1369–1377.

Marrelli

, Paduano

, Tatullo

. Human periapical cyst-mesenchymal stem cells differentiate into neuronal cells. J Dent Res, 2015; 94:843–852.

Uccelli

, Moretta

, Pistoia

. Mesenchymal stem cells in health and disease. Nat Rev Immunol, 2008; 8:726–736.

10.

Devine

, Bartholomew

, Mahmud

, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol, 2001; 29:244–255.

11.

Liechty

, MacKenzie

WTC

, Shaaban

, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med, 2000; 6:1282–1286.

12.

Simard

, Soulet

, Gowing

, Julien

, Rivest

. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron, 2006; 49:489–502.

13.

Oron

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

14.

Karu

. Ten Lectures on Basic Science of Laser Photherapy. Gragesberg, Sweden: Prima Books, 2007.

15.

Lampl

, Zivin

, Fisher

, et al. Infrared laser therapy for ischemic stroke: a new treatment strategy: results of the NeuroThera Effectiveness and Safety Trial-1 (NEST-1). Stroke, 2007; 38:1843–1849.

16.

Lapchak

, Wei

, Zivin

. Transcranial infrared laser therapy improves clinical rating scores after embolic strokes in rabbits. Stroke, 2004; 35:1985–1988.

17.

Oron

, Oron

, Chen

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

18.

De Taboada

, Yu

, El-Amouri

, et al. Transcranial laser therapy attenuates amyloid-beta peptide neuropathology in amyloid-beta protein precursor transgenic mice. J Alzheimers Dis, 2011; 23:521–535.

19.

Mvula

, Abrahamse

. Low intensity laser irradiation and growth factors influences differentiation of adipose derived stem cells into smooth muscle cells in a co-culture environment over a period of 72 hours. Interl Photoen, 2014. DOI:10.1155/2014.598793.

20.

Mvula

, Moore

, Abrahamse

. Effect of low-level laser irradiation and epidermal growth factor on adult human adipose-derived stem cells. Lasers Med Sci, 2010; 25:33–39.

21.

Oron

, Ilic

, De Taboada

, Streeter

. Ga-As (808 nm) laser irradiation enhances ATP production in human neuronal cells in culture. Photomed Laser Surg, 2007; 25:180–182.

22.

Tuby

, Maltz

, Oron

. Implantation of low-level laser irradiated mesenchymal stem cells into the infarcted rat heart is associated with reduction in infarct size and enhanced angiogenesis. Photomed Laser Surg, 2009; 27:227–233.

23.

Oron

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

24.

Tuby

, Maltz

, Oron

. 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, 2011; 43:401–409.

25.

Farfara

, Tuby

, Trudler

, et al. Low-level laser therapy ameliorates disease progression in a mouse model of Alzheimer's disease. J Mol Neurosci, 2015; 55:430–436.

26.

Farfara

, Trudler

, Segev-Amzaleg

, Galron

, Stein

, Frenkel

. gamma-Secretase component presenilin is important for microglia beta-amyloid clearance. Ann Neurol, 2011; 69:170–180.

27.

Assis

, Moretti

, Abrahao

, et al. Low-level laser therapy (808 nm) reduces inflammatory response and oxidative stress in rat tibialis anterior muscle after cryolesion. Lasers Surg Med, 2012; 44:726–735.

28.

Gavish

, Perez

, Reissma

, Gertz

. Irradiation with 780 nm diode laser attenuates inflammatory cytokines but upregulates nitric oxide in lipopolysaccharide-stimulated macrophages: implications for the prevention of aneurysm progression. Lasers Surg Med, 2008; 40:371–378.

29.

Butovsky

, Kunis

, Koronyo-Hamaoui

, Schwartz

. Selective ablation of bone marrow-derived dendritic cells increases amyloid plaques in a mouse Alzheimer's disease model. Eur J Neurosci, 2007; 26:413–416.

30.

Frenkel

, Puckett

, Petrovi

, et al. A nasal proteosome adjuvant activates microglia and prevents amyloid deposition. Ann Neurol, 2008; 63:591–601.

31.

Mildner

, Schmidt

, Nitsche

, et al. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat Neurosci, 2007; 10:1544–1553.

32.

Ilic

, Leichliter

, Streeter

, Oron

, DeTaboada

, Oron

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

33.

Tuby

, Hertzberg

, Maltz

, Oron

. Long-term safety of low-level laser therapy at different power densities and single or multiple applications to the bone marrow in mice. Photomed Laser Surg Med, 2013; 31:269–273.