ArticlePDF AvailableLiterature Review

Photostimulation of mitochondria as a treatment for retinal neurodegeneration

May 2017
Mitochondrion 36

DOI:10.1016/j.mito.2017.05.002

Project: Novel therapeutic interventions in mitochondrial optic neuropathy

Authors:

Kathy Beirne

Cardiff University

Malgorzata Rozanowska

Cardiff University

Marcela Votruba

Cardiff University

Absorption of photon energy by neuronal mitochondria leads to numerous downstream neuroprotective effects. Red and near infrared (NIR) light are associated with significantly less safety concerns than light of shorter wavelengths and they are therefore, the optimal choice for irradiating the retina. Potent neuroprotective effects have been demonstrated in various models of retinal damage, by red/NIR light, with limited data from human studies showing its ability to improve visual function. Improved neuronal mitochondrial function, increased blood flow to neural tissue, upregulation of cell survival mediators and restoration of normal microglial function have all been proposed as potential underlying mechanisms of red/NIR light.

The direct and indirect actions of red/NIR light on nitric oxide improve mitochondrial function in neurons vulnerable to degeneration, in acute neurodegenerative conditions such as TBI.

…

A proposed explanation for the observed dual effect of red/NIR light on NO in models of ischemia.

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The upregulation of ROS or RNS by red/NIR light triggers the translocation of the transcription factor NFĸB to the nucleus, enabling NFĸB to alter gene expression.

…

Figures - uploaded by Kathy Beirne

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Content uploaded by Kathy Beirne

Content may be subject to copyright.

Contents lists available at ScienceDirect

Mitochondrion

journal homepage: www.elsevier.com/locate/mito

Photostimulation of mitochondria as a treatment for retinal

neurodegeneration

Kathy Beirne

a,b

, Malgorzata Rozanowska

a,b

, Marcela Votruba

a,b,c,⁎

School of Optometry and Vision Sciences, CardiﬀUniversity, Cardiﬀ,UK

CardiﬀInstitute for Tissue Engineering and Repair, CardiﬀUniversity, Cardiﬀ,UK

CardiﬀEye Unit, University Hospital of Wales, Cardiﬀ,UK

ARTICLE INFO

Keywords:

Red light

Near infrared light

Retina

Neurodegeneration

Photostimulation

Low-level light therapy

Photobiomodulation

ABSTRACT

Absorption of photon energy by neuronal mitochondria leads to numerous downstream neuroprotective eﬀects.

Red and near infrared (NIR) light are associated with signiﬁcantly less safety concerns than light of shorter

wavelengths and they are therefore, the optimal choice for irradiating the retina. Potent neuroprotective eﬀects

have been demonstrated in various models of retinal damage, by red/NIR light, with limited data from human

studies showing its ability to improve visual function. Improved neuronal mitochondrial function, increased

blood ﬂow to neural tissue, upregulation of cell survival mediators and restoration of normal microglial function

have all been proposed as potential underlying mechanisms of red/NIR light.

1. Introduction

The therapeutic properties of light have been known since antiquity,

as far back as 1400 BCE, where it was used by Hindus to treat skin

disorders (Roelandts, 2002). The ancient Egyptians, Greeks and Romans

were also reportedly aware of the beneﬁcial eﬀects of sunlight which

they used to treat various ailments (McDonagh, 2001). The evidence for

the use of phototherapy in those time, however, is purely anecdotal. It

was not until 1903 that the therapeutic power of light gained scientiﬁc

recognition, when Niels Finsen was awarded the Nobel Prize in

medicine for the discovery of UV light as a treatment for skin

tuberculosis (lupus vulgaris)(Finsen, 1901).

Red light was, later, found to have biostimulatory eﬀects; an

unintentional discovery made by Endre Mester, in 1967, who wanted

to assess the ability of 694 nm lasers to cause carcinogenesis in mice

(Mester et al., 1971). The mice in both the light-treated and untreated

groups were shaved prior to laser exposure. The results found that the

light-treated group did not develop cancer, but more intriguingly, the

hair grew back on the laser treated mice at a faster rate than the

untreated group.

In more recent times, there has been a surge in the use of red and

near infrared (NIR) lasers and LEDs in clinical and preclinical research

(Desmet et al., 2006). As red and NIR light have relatively long

wavelengths, they have the advantage of a greater penetration depth

over shorter wavelengths, making them an ideal choice for the

treatment of neural tissue (Hartwig and Van Veen, 1979). In addition

to light being able to penetrate into the tissue of interest, another

requirement is that the photon energy corresponds to the absorption

characteristics of the chromophores responsible for triggering the

beneﬁcial eﬀects upon photoexcitation. It appears that red and NIR

light correspond to the absorption maxima of such chromophores as

will be discussed later. For various reasons, LEDs are most commonly

used as the light source in these studies. Most importantly, red/NIR LED

therapy has been approved for use in humans and has been deemed as a

non-signiﬁcant risk by the U.S. Food and Drug Administration.

Although shorter wavelengths of visible light and UV light are also

employed for therapeutic purposes, their safety for use in humans,

especially for the eye, is less clear (Rozanowska et al., 2009;

Rozanowska, 2012). With the ultimate objective of exploring the

eﬃcacy of phototherapy as a treatment for neurodegeneration in the

human retina, this review will focus only on the use of wavelengths that

are least likely to cause adverse eﬀects, that is, red and NIR light

(Barolet, 2008).

http://dx.doi.org/10.1016/j.mito.2017.05.002

Received 15 July 2016; Received in revised form 15 February 2017; Accepted 8 May 2017

⁎

Corresponding author at: School of Optometry and Vision Sciences, CardiﬀUniversity, Cardiﬀ, UK.

E-mail addresses: BeirneK@cardiﬀ.ac.uk (K. Beirne), RozanowskaMB@cardiﬀ.ac.uk (M. Rozanowska), VotrubaM@cardiﬀ.ac.uk (M. Votruba).

Abbreviations: NIR, near infrared; LED, light emitting diode; FDA, the U.S. Food and Drug Administration; COX, cytochrome coxidase; RGC, retinal ganglion cell; ERG,

electroretinogram; NO, nitric oxide; ROS, reactive oxygen species; RNS, reactive nitrogen species; NF, nuclear factor; IL, interleukin

Mitochondrion 36 (2017) 85–95

Available online 09 May 2017

MARK

2. The potential of red/NIR light as a treatment for

neurodegeneration

2.1. Evidence from in vitro studies

Red and NIR light have been shown to provide protection against

the deleterious eﬀects of mitochondrial electron transport chain

inhibitors and excitotoxic cell death in neurons in vitro (Wong-Riley

et al., 2005; Ying et al., 2008; Huang et al., 2014). Since impaired

mitochondrial function and excitotoxicity are common causes of cell

death in neurodegenerative conditions, the ability of red/NIR light to

protect against these challenges in vitro has emphasized the potential of

this therapy in various neurodegenerative conditions.

2.2. The eﬀects of red/NIR light in models of neurodegeneration

Red/NIR light therapy has shown great potential in the treatment of

acute neurodegenerative conditions, showing neuroprotective eﬀects in

rodent models of spinal cord injury, traumatic brain injury and stroke

(Byrnes et al., 2005; Wu et al., 2012; Xuan et al., 2015; Dong et al.,

2015; Oron et al., 2006; Giacci et al., 2014).

Furthermore, red light has been shown to have beneﬁcial eﬀects in

animal models of some of the most prevalent neurodegenerative

diseases. A reduction in cell loss and other markers of disease severity

was seen with red/NIR light treatment, in rodent models of multiple

sclerosis, Alzheimer's and Parkinson's disease (Muili et al., 2012, 2013;

Purushothuman et al., 2013, 2015; Oueslati et al., 2015; Johnstone

et al., 2014; Peoples et al., 2012; Shaw et al., 2010).

2.3. The potential of red/NIR light as a successful treatment for

neurodegeneration in humans

While transcranial red/NIR light therapy is yielding remarkable

results in numerous rodent models of neurodegeneration, the real

question is how well these results will translate when applying this

therapy to human patients.

Interestingly, in a neurotoxin-induced monkey model of Parkinson's

disease, 670 nm light was delivered directly to the macaque midbrain

using an implanted optical ﬁbre which was activated over the period of

time of 5–7 days when the neurotoxin precursor, 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine (MPTP), was injected (Darlot et al., 2016).

The study found a reduction in clinically-assessed behavioural impair-

ment with this method of red light delivery in this primate model as

well as neuroprotection to the dopaminergic neurons of the substantia

nigra. Although a more invasive method of delivery than transcranial

red light treatment, no major adverse eﬀects were observed following

surgical implantation of the optical ﬁbre. However, it would have been

of great interest if the eﬀects of transcranial light delivery were also

tested in this model, for comparison.

3. Red light treatment in retinal degenerative diseases

Since the retina is an extension of the CNS, the neuroprotective

eﬀects of red/NIR light, as discussed above, should also be observed in

this tissue. In fact, irradiating the retina with red or NIR light seems

more likely to be successful as a non-invasive treatment for human

patients as the issue of tissue penetration is avoided.

3.1. The safety of light treatment on the retina

The greatest concern arising when aspiring to use red light therapy

to treat retinal degeneration, is the potential retinal damage that may

occur upon direct exposure of the retina to light with high levels of

irradiance. The dangers of high levels of irradiance on the retina is

highlighted in a study on anesthetised monkeys (Friedman and

Kuwabara, 1968). It was found that white light with a retinal irradiance

of 270 mW/cm

caused irreversible damage to the photoreceptors and

retinal pigment epithelium. White light is made up of light of all

wavelengths in the visible light spectrum, with light of shorter

wavelengths and higher frequencies having a greater damaging eﬀect

on photoreceptors. Blue light, with a relatively short wavelength, was

found to cause irreversible damage to S cones (Harwerth and Sperling,

1975). While exposure to green and red light caused damage to M and L

cones, respectively, the damage to these cones was reversible, with a

full recovery of function seen after a few weeks. More recent studies on

macaque monkey, however, have demonstrated that yellow light of

568 nm wavelength can cause retinal damage manifested as disruption

of the retinal pigment epithelium at the dose below the Maximal

Permissible Exposure established by the American National Standard

Institute's (ANSI) as a standard for the safe use of lasers (Hunter et al.,

2012).

Albeit transient and less severe than light of shorter wavelengths,

damage to L cones upon exposures to high levels of red light would be a

cause for concern when considering red light as a treatment for retinal

degeneration. This concern has been addressed with numerous in vivo

studies. These studies have shown that therapeutic eﬀects were

achieved, in the absence of retinal damage, when rodent retinas were

exposed to 670 nm light with a therapeutically eﬀective irradiance and

exposure times (Albarracin et al., 2011, 2013; Giacci et al., 2014).

Further, this included irradiance of 60 mW/cm

which is the highest

irradiance level found in studies of in vivo models of retinal degenera-

tion where positive results were achieved using 670 nm light. This

demonstrates the safety of using 670 nm light as a treatment for retinal

degeneration.

In addition to photoreceptor damage, the possibility of photother-

mal damage to the retina and surrounding ocular structures evokes

further concern when considering using light to treat retinal degenera-

tion (Youssef et al., 2011). Comparing the eﬀects of green, red and NIR

laser light exposure on the temperature rise in the human choroid, it

was found that the longer wavelengths led to a smaller degree of

choroidal heating, due to the decrease in absorption by melanin with

increasing wavelength (Vogel and Birngruber, 1992). The variation in

choroidal heating between green and red wavelengths was minor

compared with the diﬀerence between green and NIR wavelengths.

Still the question remains as to which would be the optimal

wavelength for use as a neuroprotective agent in the retina.

Addressing this, the eﬃcacy of red and NIR light was compared in a

model of partial optic nerve transection (Giacci et al., 2014). It was

found that although protective eﬀects were seen in retinas treated with

both red and NIR light, 670 nm light was more eﬀective in improving

visual function compared with 830 nm light. However, in a rat model of

light induced retinal degeneration protective eﬀects were observed

with 670 nm light treatment, but no protection was seen with 830 nm

light (Giacci et al., 2014). It is therefore not surprising that most studies

testing the eﬀectiveness of phototherapy on neurodegeneration in the

retina use red light at 670 nm.

3.2. 670 nm light therapy in models of photoreceptor damage

As discussed above, exposing the retina to bright light can cause

photoreceptor damage, an event that can occur with excessive sunlight

exposure or accidental exposure to high intensity artiﬁcial light sources.

Models of light induced photoreceptor damage are also used to simulate

retinal degenerative diseases, involving photoreceptor speciﬁc death.

Emphasizing the vast and diverse eﬀects of light on biological tissue,

irradiating the retina with red light provided protection against the

structural damage to the outer retina and loss in photoreceptor function

in a rat model of light induced photoreceptor degeneration (Albarracin

et al., 2011). Methanol can also induce damaging eﬀects on the retina

causing photoreceptor toxicity due to the ability of its metabolite,

formic acid, to inhibit cytochrome coxidase, the terminal enzyme of the

electron transport chain. In a rat model of methanol induced retinal

K. Beirne et al. Mitochondrion 36 (2017) 85–95

toxicity, red light treatment brought about a signiﬁcant recovery of rod

and cone mediated function, in addition to preventing methanol

induced changes to outer retinal morphology (Eells et al., 2003). These

studies show the ability of red light to protect against loss of photo-

receptor function, an event that would cause severe visual impairment

and would otherwise be irreversible.

3.3. The eﬀects of red light on inﬂammation in the outer retina

Inﬂammation in the retina has been implicated in many retinal

diseases including age related macular degeneration (AMD) and

diabetic retinopathy, and as such, looking at ways to alleviate

inﬂammation in these conditions is thought to reduce the severity of

symptoms associated with these diseases (Whitcup et al., 2013). Also,

an upregulation in inﬂammatory proteins has been observed following

light damage in rats, demonstrating pathological features similar to

“dry”AMD (Rutar et al., 2012). In addition to its ability to protect

against light induced photoreceptor damage, red light treatment was

found to reduce the complement propagation that occurs in the retina

following light damage. Red light treatment was also tested in an aged

genetic mouse model of AMD, the complement factor H knockout,

which, likewise, presents with reduced retinal function and increased

inﬂammation (Begum et al., 2013). In contrast to most studies with

red/NIR light, where the light source was held directly in front of the

animal, the environmental light was supplemented with red light, for

this study. Even though the red light exposure was indirect, the levels

reaching the retina were suﬃcient to reduce inﬂammation in the outer

retina, in this model.

In addition to inﬂammation associated with disease pathology,

inﬂammation increases in the retina with age. Red light was eﬀective

in reducing proinﬂammatory cytokines and a chronic marker of

inﬂammation in the aged mouse retina, demonstrating the potential

of red light to also alleviate the visual decline associated with normal

aging (Kokkinopoulos et al., 2013). These studies draw attention to the

ability of red light to produce anti-inﬂammatory eﬀects associated with

outer retinal degeneration, whether by induced damage, disease

pathology or normal aging.

3.4. The eﬀects of red light on neurodegeneration in the inner retina

Retinal ganglion cell (RGC) death and optic nerve degeneration are

hallmarks of optic neuropathy, a frequent cause of vision loss, of which

the causes are many. Red light therapy has been trialled in a rat model

of diabetic retinopathy and positive outcomes have been reported.

There was a signiﬁcant reduction in the diabetes induced RGC death

and a 50% improvement in the diabetes-induced reduction in ERG

amplitude with exposure to red light (Tang et al., 2013). Highlighting

the beneﬁcial eﬀects of red light on the retinal vasculature, red light

also prevented the diabetes-induced increase in leukostasis in the

retinal vasculature, an event which is implicated in the pathogenesis

of diabetic retinopathy. Further, the diabetes-induced increase in

retinal expression of an adhesion molecule, essential for leukostasis,

ICAM-1, was also prevented with red light. Similarly, in a model of

secondary degeneration of RGCs, resulting from traumatic injury to the

optic nerve, red light was found to be protective (Fitzgerald et al.,

2010). The secondary damage, following partial optic nerve transec-

tion, normally leads to further loss of RGCs and visual function;

however, normal visual function was restored with red light treatment

(Fitzgerald et al., 2010). Furthermore, treatment with 670 nm light in a

rat model of partial optic nerve transection, resulted in improved

vision, 7 days post injury (Giacci et al., 2014). Additionally, dendro-

pathy of retinal ganglion cells, an event found to be associated with

visual loss in experimental models of glaucoma and autosomal domi-

nant optic atrophy, was partially prevented, in an axotomy model of

neurodegeneration, with red light treatment (Beirne et al., 2016).

Protection against RGC and optic nerve degeneration, arising from

diﬀerent conditions, appears to be possible with red light treatment.

Since RGC dysfunction and optic nerve degeneration are common

features among numerous other types of optic neuropathies red light

has the potential to provide protection in these conditions also.

However, the successful outcomes achieved in the discussed experi-

mental models were seen when red light was administered immediately

after induced optic nerve injury, a treatment strategy that would be

unachievable in a real life clinical setting. Although the therapeutic

window of opportunity for red/NIR light therapy in models of traumatic

optic neuropathy has not been explored, it has been assessed in other

CNS injury models. NIR light therapy improved motor function in a

rabbit model of embolic stroke when treatment was administered

6 hour post-embolization but was ineﬀective when administered

12 hour post embolization (Lapchak et al., 2007). The ﬁndings show

that neuroprotection can be achieved when red/NIR light is adminis-

tered for up to 6 h after the induced injury, showing the therapy to be

applicable to a clinical setting. Other studies on animals demonstrated

eﬀectiveness of near-infrared light in a mouse model of traumatic brain

injury when administered 4 h after injury, with additional treatments

administered at one day and two days post injury (Xuan et al., 2016;

Thunshelle and Hamblin, 2016). The transcranial treatment upregu-

lated brain-derived neurotrophic factor (BDNF), improved neurological

functions, reduced the size of the lesion, stimulated formation of new

neurons and synaptogenesis. There is also a growing body of evidence

suggesting that people aﬀected by chronic traumatic brain injury or

after stroke can beneﬁt from transcranial irradiation with red/near-

infrared light (Naeser et al., 2016; Hamblin et al., 2016).

3.5. Red/NIR light as a therapy for patients with retinal disease

The protective eﬀects seen with red light treatment, in the absence

of adverse eﬀects, in numerous in vivo models of retinal degeneration,

strongly suggest this non-invasive treatment should be trialled in

patients with retinal degeneration. Progress to this end has commenced

with trials of red and NIR light therapy yielding promising results in

patients with AMD. In one such study, a brief exposure of NIR light

(780 nm), from a semiconductor laser diode, to AMD patients, twice per

week for two weeks, resulted in a signiﬁcant improvement in their

visual acuity (Ivandic and Ivandic, 2008). This improvement in vision

was seen in patients with both wet and dry AMD and was maintained

for 3–36 months after treatment. Moreover, no adverse eﬀects of the

treatment were seen. In this study, the laser was applied transconjunc-

tivally, to the macula, when the eye was in adduction. In another study

looking at the eﬀects of photobiomodulation on patients with dry AMD,

the retina was irradiated through the pupil with red light (670 nm),

from the FDA approved Warp 10 LED light source, 3 times per week for

6 weeks (Merry et al., 2013). The visual acuity and contrast sensitivity

remained signiﬁcantly improved for 12 months after treatment; how-

ever, the improvement in visual acuity began to decline after 4 months.

The results provide vital information on the time at which patients may

beneﬁt from re-treatment, in addition to providing pilot data on the

safety and eﬀectiveness of 670 nm light from an LED source. Red/NIR

light has also been trialled in diabetic macular edema and Leber's

hereditary optic neuropathy, however, there are no results available

from these studies. A summary of all the studies using red/NIR light as a

treatment intervention in conditions associated with neurodegeneration

in the retina is available in Table 1. From all the studies listed, red/NIR

light is showing the greatest potential as a treatment for AMD. Since

positive outcomes have been observed in patients with AMD, this paves

the way for the application of this therapy in other retinal degenerative

conditions, particularly those where positive outcomes have been seen

in preclinical studies.

4. The eﬀects of red/NIR light on mitochondrial dysfunction

Although the therapeutic beneﬁts of red/NIR light therapy have

K. Beirne et al. Mitochondrion 36 (2017) 85–95

been demonstrated in a number of diﬀerent disease models, in addition

to AMD patients, the underlying molecular mechanisms are less well

understood (Desmet et al., 2006). The question is no longer whether or

not light has biological eﬀects, it is rather how these eﬀects are

mediated at a cellular and molecular level (Hamblin and Demidova,

2006).

4.1. Cytochrome c oxidase: the photoacceptor for red/NIR light

Endeavors to uncover the underlying molecular mechanisms suggest

a major role for cytochrome coxidase (COX) which is the terminal

enzyme of the electron transport chain, transferring electrons from

cytochrome cto molecular oxygen (Chung et al., 2012; Karu, 1999).

COX is a large multicomponent protein, containing two copper

centers (Cu

and Cu

) and two heme iron containing centers (heme a

and heme a3), which absorbs photons in the red to NIR region of the

electromagnetic spectrum (Karu, 1999). These transition metals are

also the intermediate redox sites in the electron transfer pathway from

cytochrome cto oxygen, a process which is coupled to the pumping of

protons across the inner mitochondrial membrane. The electrons pass

from cytochrome cto Cu

then passed to heme a, from heme ato heme

a3-Cu

and ﬁnally to molecular oxygen.

4.2. The absorption of photons by photoacceptors in COX

One theory proposed to explain how photon energy is absorbed by

COX, centers on its heme molecules (Zielke, 2014). Heme is comprised

of a porphyrin ring with an iron atom at its center that can continuously

switch its oxidation states between ferrous (Fe

) and ferric (Fe

)by

accepting or donating an electron. The porphyrin ring is made up of

four pyrrole rings that are connected through their carbon atoms via π

bonds. The electrons in these πbonds are delocalised, moving back and

forth from one conﬁguration to another, creating resonance. Electrons,

like photons, have a dual nature, behaving like particles or electro-

magnetic waves, thereby creating a resonating electromagnetic cloud in

the porphyrin ring. Photons with similar wavelengths are absorbed by

this cloud, increasing its energy. The energy from these photons causes

photoexcitation of electrons of Fe

, bringing them to an unstable

higher energy level. Upon absorption of suﬃcient energy, these

electrons are released from the orbitals of Fe

causing the oxidation

of Fe

to Fe

. The oxidised iron atom can then accept electrons from

cytochrome c, thus increasing the electron ﬂux through the electron

transport chain.

Experimental evidence shows that red/NIR light has the ability to

upregulate the enzymatic activity of complex IV, increase the mito-

chondrial membrane potential and increase ATP production (Begum

et al., 2013; Kokkinopoulos et al., 2013; Hamblin and Demidova, 2006;

Karu, 2008; Ferraresi et al., 2015a,b; Tina Karu et al., 2013). These

mitochondrial speciﬁceﬀects of photobiomodulation may oﬀer a

partial explanation for its beneﬁcial eﬀects in neurodegenerative

diseases associated with mitochondrial dysfunction. However, in genet-

ic or toxin induced models of Parkinson's disease, where loss of

dopaminergic cells was triggered by complex I dysfunction, neuropro-

tective eﬀects were seen with red/NIR light treatment. The absorption

of photon energy by the heme group in complex IV may explain how

photobiomodulation can increase ATP production when complex IV is

inhibited but fails to explain how this eﬀect can be achieved in models

with complex I inhibition.

It has been hypothesised by Zielke et al. that the electrons released

in this oxidation process are free to reduce NAD

and FAD, providing

substrates for complex I and II, respectively, creating a closed circuit of

electron transfer (Zielke, 2014). In the case of aberrant functioning of

COX the ﬂux of electrons through this closed circuit would maintain the

proton pumping functions of complex I and III, thereby maintaining the

electrochemical gradient across the mitochondrial membrane required

for ATP synthesis. However, since oxygen is very electronegative it

would be most likely that the released electrons would be readily

accepted by the oxygen molecule bound to the reduced heme a3-Cu

component of COX, reducing it to water. In the event of aberrant COX

activity the released electron would most likely react with unbound

oxygen molecules, forming reactive oxygen species. Therefore, how

red/NIR provides neuroprotection against complex I dysfunction is not

explained by its direct action on the COX.

5. The nitric oxide theory of red/NIR light therapy

5.1. The role of nitric oxide in mitochondrial respiration

Nitric oxide (·NO) has important roles in the regulation of blood

pressure and vasculature tone, however, excessive ·NO production, as

seen in neurodegenerative diseases, can cause impairment of mitochon-

drial respiration and apoptosis (Zhao, 2005). Mitochondria harbor

nitric oxide synthase (NOS) to produce NO, which they use to hinder

respiration, as an intrinsic mechanism to prevent oxygen from reaching

precariously low levels (Barolet, 2008). ·NO, at low concentrations,

competes with oxygen to bind to the reduced heme a3-Cu

component

of COX (Chung et al., 2012). This prevents COX from reducing

molecular oxygen, thus impairing the proton pumping abilities of the

enzyme and essentially the energy production ability of the mitochon-

dria. Additionally, ·NO was found to cause inhibition of complex I, II

and IV-dependent respiration in mitochondrial suspensions where the

inhibitory action of ·NO was found to be more profound at lower oxygen

tensions (Cassina and Radi, 1996). Interestingly, this inhibitory action

of ·NO was overcome by reoxygenation of mitochondrial suspensions

for a mere 10 s, resulting in complete recovery of complex I and IV

dependent oxygen consumption and a 50% recovery of complex II

dependent respiration. This shows that the inhibitory actions of ·NO are

almost completely reversible upon restoration of normal oxygen levels.

Table 1

A summary of the completed, planned and terminated clinical trials, using red/NIR light as a treatment intervention in conditions associated with retinal neurodegeneration.

Responsible party Last veriﬁed on

ClinicalTrials.gov

Condition Duration of

treatment period

Number of

patients

Duration of improved

vision after treatment

Status

Harry T Whelan 01/09/2013 Diabetic macular edema 3 months 20 No results available Completed

Ivandic and Ivandic

(2008)

N/A exudative and non-

exudative AMD

2 weeks 203 Up to 36 months Completed and published

(Ivandic and Ivandic, 2008)

Merry et al. (2013) 01/11/2011 Non-exudative age-related

macular degeneration

6 weeks 9 Up to 12 months Completed and published

(Merry et al., 2013)

Merry et al. (2016) N/A Non-exudative age-related

macular degeneration

3 weeks 24 3 months Completed and published

(Merry, 2016)

University of Sydney 01/06/2014 Diabetic retinopathy 4 weeks N/A N/A Planned

LumiThera, Inc. 01/04/2016 Age-related macular

degeneration

3 weeks 30 N/A Planned

Harry T Whelan 01/09/2014 Leber's hereditary optic

neuropathy

3 months 4 N/A Terminated (0/4 patients

completed the study)

K. Beirne et al. Mitochondrion 36 (2017) 85–95

Therefore, this intrinsic mechanism would protect a tissue if the

depleted oxygen supply were temporary, by putting the mitochondria

in a state of conservation until the return of normal oxygen levels.

However, prolonged inhibition of respiration would deplete ATP levels

and bring about cell death (Kalogeris et al., 2012).

5.2. The eﬀects of red/NIR light on ·NO-mediated mitochondrial

dysfunction

Red/NIR light has been proposed to inﬂuence the photodissociation

of ·NO from COX, thereby allowing oxygen to reclaim its binding site,

permitting the ATP production process to resume (Chung et al., 2012).

This would be most beneﬁcial in pathological situations where ·NO

levels are higher than normal physiological amounts, favouring the

binding of ·NO rather than O

to COX. Experiments have shown that

irradiating cells with red/NIR light increases COX activity in normal

healthy neurons and restores the activity in COX inhibited neurons

(Wong-Riley et al., 2005). However, the increase in COX activity

appears to be mediated by an increase in the expression of the proteins

in the COX complex, suggesting that a mechanism, additional to the

disinhibition of COX, may also be involved (Lim et al., 2010).

Furthermore, exposure of cells to an irreversible COX inhibitor was

overcome by NIR light treatment, providing further support to the claim

that restoration of activity is mediated by upregulating the expression

of COX proteins (Wong-Riley et al., 2005).

An indirect eﬀect of red/NIR light on COX activity helps explain

how beneﬁcial eﬀects are also seen in neurodegenerative conditions

that are associated with impairment of other electron transport chain

complexes. The absorption of photon energy from red/NIR light by COX

may indeed have direct eﬀects on the enzyme itself, however, this

initial event may trigger further downstream events, which may have

more far-reaching eﬀects. As ·NO is widely known for its function as an

intercellular signaling molecule, the release of ·NO upon red/NIR light

exposure would increase its bioavailability allowing it to function as a

signaling molecule (Lohr et al., 2009). It has been suggested that ·NO

intracellular signaling could play a role in the upregulation of COX

proteins upon red/NIR light exposure. Intracellular signaling from the

mitochondria to the nucleus may be triggered by other byproducts of

mitochondrial respiration, levels of which may be altered by red/NIR

light, therefore, will be discussed in more detail later in this review.

5.3. The indirect eﬀects of red/NIR light on mitochondrial dysfunction

The ﬁndings by Cassina and Radi suggest that increasing the

delivery of oxygen to the mitochondria, as would occur with increased

blood ﬂow to the tissue, would improve mitochondrial function in

tissues with ·NO-mediated mitochondrial electron transport chain

inhibition (Cassina and Radi, 1996). It has been found that the exposure

of blood vessels to red light from an LED source can induce photo-

relaxation of blood vessels, an event that would increase blood ﬂow

and, in turn, oxygen delivery to the irradiated tissue (Plass et al., 2012).

Exposure of porcine coronary arteries to red light caused their

vasodilation, as measured by wall tension in the exposed vessels. Since

nitric oxide is known to have a primary role in the regulation of

vasculature tone, and NOS is activated upon absorption of visible light,

the vasodilation eﬀect seen upon red light exposure is thought to be

mediated through nitric oxide (Samoilova et al., 2008). Additionally,

red/NIR light can trigger the photodissociation of ·NO from nitrosyl

hemoglobin and nitrosyl myoglobin (Lohr et al., 2009). The ·NO

released from hemoglobin in the blood would contribute to the

vasodilation eﬀects of red/NIR light. Depending on the physiological

situation, photobiomodulation can either reduce or increase ·NO levels,

however, the molecular events determining whether the eﬀect will be

an inhibitory or a stimulatory one are yet to be identiﬁed (Gavish et al.,

2008). An increase in ·NO upon red light exposure would provide

beneﬁcial eﬀects in conditions such as TBI, where increased cerebral

blood ﬂow could increase mitochondrial function in hypoxic cells

(Fig. 1)(Naeser et al., 2014). As mentioned earlier in the review,

hypoxia can trigger inhibition of respiration through the binding of ·NO

to COX. The photodissociation of ·NO from COX in hypoxic tissue may

not cause a signiﬁcant improvement in mitochondrial function since

there would be limited oxygen available to reclaim the binding site on

Fig. 1. The direct and indirect actions of red/NIR light on nitric oxide improve mitochondrial function in neurons vulnerable to degeneration, in acute neurodegenerative conditions such

as TBI.

K. Beirne et al. Mitochondrion 36 (2017) 85–95

COX. However, if combined with an increase in cerebral blood ﬂow, the

associated increase in oxygen levels would lead to a more substantial

improvement in mitochondrial function. This proposes a partial ex-

planation for the neuroprotective eﬀects seen upon red/NIR light

exposure.

5.4. The eﬀects of red/NIR light on cellular function

As mentioned above, the ability of red/NIR light to increase free ·NO

can have beneﬁcial eﬀects in cells where mitochondria are dysfunc-

tional. Providing further support to this theory, it was found that red/

NIR light was protective against hypoxia and re-oxygenation injury in

cultured cardiomyocytes. The observed protection was dependent on an

increase in ·NO as the protective eﬀects were abolished in the presence

of ·NO scavengers (Zhang et al., 2009). Further, it was observed that the

increase in ·NO seen upon red/NIR exposure was partially prevented by

the non-selective inhibition of all isoforms of NOS. Of note, there are

three isoforms of NOS: Neuronal NOS (nNOS), which is expressed in the

central nervous system and plays a role in synaptic plasticity and

central regulation of blood pressure, endothelial NOS (eNOS), which is

mostly expressed in endothelial cells and primarily functions in

controlling blood pressure, and inducible NOS (iNOS), which can be

expressed in many cell types in response to cytokines and other agents

to generate large amounts of ·NO (Förstermann and Sessa, 2012). The

ﬁndings show that the increase in ·NO by red/NIR light is mediated in

part by its action on NOS, however, the exact isoform of NOS

responsible for the protective eﬀects has not been determined. The

source of the remaining NO could be that which is released during the

photodissociation of ·NO from COX in the mitochondria as discussed

above.

This in vitro model of cardiac ischemia provides useful insight into

how red/NIR light mediates its eﬀects via ·NO in ischemic conditions.

Since the experiment was done in vitro, the observed protective eﬀects

most likely arose from the local eﬀects of an increase in intracellular

·NO rather than an indirect eﬀect of ·NO by increasing blood ﬂow to the

ischemic tissue. Although reperfusion is essential to limit cell death

after hypoxia, paradoxically, this event itself causes further cell death

due to excess ROS production (Keszler et al., 2014). The increase in free

·NO upon red/NIR light exposure would increase its availability to bind

to COX and cause the reversible S-nitrosation of complex I, events

which would slow down the reactivation of the electron transport chain

during the crucial initial stages of reperfusion (Chouchani et al., 2013).

In this particular pathological situation red/NIR light, administered

upon reoxygenation, could reduce the harmful levels of ROS produced

during reperfusion injury by reversibly inhibiting the electron transport

chain.

Contrastingly, in an in vivo model of cerebral ischemia, where excess

·NO production is said to be associated with neurotoxic eﬀects, red/NIR

light has been shown to have the ability to reduce the levels of ·NO by

down-regulating the activity and expression of all isoforms of NOS

(Leung et al., 2002). Both studies focus on ·NO to explore the under-

lying mechanism responsible for the protective eﬀect of red/NIR light

in models of ischemia, yet, in these examples, the eﬀects on ·NO were

found to be conﬂicting. The respective increase and decrease in ·NO

levels seen upon red/NIR light irradiation in the discussed models, was

dependent on the respective activation and inhibition of NOS. Although

theories have been proposed to explain the increase in NOS activity in

response to red/NIR light irradiation, how red/NIR light inhibits NOS

activity is less clear. Since the expression of the three isoforms of NOS

showed a similar trend to the speciﬁc activities of the NOS enzymes in

response to red/NIR light, red/NIR light must be somehow suppressing

the expression of NOS, but the mechanism responsible for this eﬀect is

unknown (Leung et al., 2002).

This observed dual eﬀect of red/NIR light on intracellular ·NO levels

has great relevance in the ﬁeld of neurodegenerative conditions. ·NO at

physiological amounts confers neuroprotection, however, if produced

in excess, ·NO has neurotoxic eﬀects (Calabrese et al., 2007). In the in

vivo model of cerebral ischemia mentioned above the light was

administered immediately after middle cerebral artery occlusion but

the levels of NOS activity were not measured until 4 days post injury,

the time at which the NOS levels peaked before returning to pre-injury

levels. In the in vitro model of cardiac ischemia the light was also

administered immediately after hypoxia but the ·NO levels were

measured after just 2 h of reoxygenation. It is possible, therefore, that

red/NIR light triggers an initial increase in ·NO levels, suﬃcient to

reduce ROS production and bring about the observed cytoprotective

eﬀects. Furthermore, as the cell is then in a state of elevated ·NO levels

and reduced ROS levels this may be suﬃcient to switch oﬀthe

Fig. 2. A proposed explanation for the observed dual eﬀect of red/NIR light on NO in models of ischemia.

K. Beirne et al. Mitochondrion 36 (2017) 85–95

endogenous trigger that induces the increased expression of NOS and

the subsequent delayed surge in ·NO levels, which only contribute to the

toxic eﬀects at that late stage of ischemia. However, as shown in Fig. 2,

low levels of ·NO are produced during the early stages of ischemia to

induce neuroprotective eﬀects in the absence of red/NIR light, yet an

increase in ·NO is responsible for the neuroprotective eﬀects achieved

with red/NIR light in the in vitro model of ischemia. Therefore, how

does the ·NO produced by red/NIR light provide further neuroprotec-

tive eﬀects in the early stages of ischemia? Also unanswered is how the

increase in ·NO by red/NIR light and the associated reduction in ROS

production would downregulate the delayed surge in NO production in

the later stages of ischemia when the ·NO produced in the absence of

red/NIR light fails to do so.

There is much evidence to show that ·NO acts as a neuroprotective

agent through its various cellular eﬀects. One such eﬀect is the

induction of the signaling molecule cyclic guanosine 3′,5′-monopho-

sphate (cGMP), a molecule with a key role in vasodilation and

mitochondrial biogenesis (Tengan et al., 2012). Mitochondrial biogen-

esis has been found to occur in response to red/NIR light irradiation

(Nguyen et al., 2014). Diseases with a mitochondrial origin such as

Leber's hereditary optic neuropathy, retinitis pigmentosa and autosomal

dominant optic atrophy would beneﬁt from the associated increase in

mitochondrial biogenesis, as a way of supporting neuronal survival.

5.5. The eﬀects of reactive nitrogen species on mitochondrial function

There is much evidence to show that ·NO, at higher doses, is toxic to

neurons; however, this NO-mediated toxicity is not produced by ·NO

alone, but by the formation of reactive nitrogen species (RNS) (Lipton

et al., 1993). As a by-product of mitochondrial respiration the super-

oxide radical anion (O

−

) is formed when electrons from complex I or

III are transferred to oxygen molecules instead of their respective

substrates: ubiquinone and cytochrome c(Lenaz, 2001). If the amount

of superoxide produced in the cell increases to a level that would

exceeds the antioxidant capacity of the cell, oxidative stress will result.

In that pro-oxidant state, the production of ·NO can cause the genera-

tion of additional cytotoxic compounds. The reaction of NO with the

superoxide radical anion (O

−

) leads to the generation of the powerful

oxidant peroxynitrite (ONOO

−

), which causes detrimental eﬀects in the

cell, through its interactions with lipids, proteins and DNA (Lipton

et al., 1993).

Nitric oxide, superoxide and peroxynitrite are often generated in

excess during inﬂammatory and pathological conditions, contributing

to the associated toxic eﬀects (Rubbo et al., 1994). Peroxynitrite can

induce mitochondrial dysfunction and cell death in neurons by its

ability to inhibit many mitochondrial proteins including complex I, II

and IV, and ATP synthase. It can also increase mitochondrial proton

permeability, an eﬀect which may be caused by lipid peroxidation

(Brown and Borutaite, 2004). Impairment of normal mitochondrial

function causes depletion of ATP and generation of free radicals,

causing cellular dysfunction and further oxidative stress. Peroxynitrite

has been found to be involved in the pathogenesis of many neurode-

generative disorders (Torreilles et al., 1999). Since the formation of

peroxynitrite depends on the availability of ·NO, inhibition of nitric

oxide synthase activity, as was found to occur in cerebral ischemic-rats

irradiated with red light, may reduce the amount of peroxynitrite

produced and the associated deleterious eﬀects in neurodegenerative

disorders (Leung et al., 2002).

6. Red/NIR light in the mitochondrial signaling pathway

Causing further controversy in the eﬀorts to uncover the underlying

molecular mechanism of red/NIR light therapy, is the eﬀect that it has

on ROS and RNS production. It is also uncertain whether an increase or

a decrease in these molecules, in the cell, in response to red/NIR light

exposure would be the most therapeutically beneﬁcial. In some

physiological situations red light mediates its therapeutic eﬀects by

increasing levels of free radicals but in other circumstances by reducing

the levels of free radicals. Consequently, a further look at the molecular

eﬀects of free radicals in neurodegeneration is required to uncover the

underlying mechanism of red/NIR therapy.

6.1. The role of ROS and RNS in neurodegeneration

Postmortem analysis of the brains of patients with various neuro-

degenerative diseases shows an increase in ROS and RNS in the aﬀected

brain regions (Andersen, 2004; Aslan and Ozben, 2004). It is known

that these reactive species can cause oxidative and nitrative damage to

cellular components thereby having toxic eﬀects on the cell (Tafur and

Mills, 2008). It may be deduced from this association that the increase

in ROS and RNS is contributing to the cellular death and that an

antioxidant may be beneﬁcial in such conditions. Since red/NIR light

has been found to be protective in models of such conditions it could,

therefore, be possible that the therapeutic beneﬁts of red/NIR light

could be due, in part, to an antioxidant eﬀect. One possible mechanism

of providing the antioxidant protection is the photodissociation of ·NO,

which at low concentrations can exert an antioxidant eﬀect (Niziolek

et al., 2003a,b, 2005, 2006).

Because red/NIR light has been found to increase the activity of the

electron transport chain, and ROS/RNS production is a byproduct of

such activity, sometimes it is presumed that red/NIR light increases

ROS production. It can be argued, however, that by removing NO-

mediated inhibition of COX, the electron ﬂow is restored and therefore

the likelihood of donating an electron from complex I or III to oxygen

(which results in generation of superoxide radical anion) is reduced

(Lenaz, 2001). Thus by enabling the electron ﬂow in the electron

transport chain, the risk of formation of superoxide is decreased.

Investigation into the eﬀects of red/NIR light on ROS production by

various groups provides inconsistent results as some found a reduction

in ROS upon irradiation while others found that ROS was, in fact,

upregulated (Tafur and Mills, 2008). Regardless of the eﬀect of red/NIR

light on ROS levels, it is unclear whether an antioxidant or pro-oxidant

eﬀect would be most beneﬁcial when employing red/NIR light as a

neuroprotective agent. Regulated ROS production could trigger signal-

ing pathways involved in cell protection, but unregulated ROS produc-

tion could result in cellular damage and cell death (Zorov et al., 2014).

In addition, when ROS levels are too low, this also has detrimental

eﬀects for the cell. The concept that lower, non-toxic levels of ROS are

essential for promoting cell survival by inducing an adaptive responses

is called mitochondrial hormesis or mitohormesis (Ristow and

Schmeisser, 2014). Exposure to red/NIR light may trigger a transient

increase in ROS levels, suﬃcient to induce this adaptive response and

provide neuroprotective eﬀects. This explanation seems plausible in

situations where light is administered as a pre-treatment as the cells

could employ this adaptive response to protect against a subsequent

injury-induced increase in ROS levels. However, if the cell is already in

a state of elevated ROS levels, as in many pathological situations, it is

unclear as to how an additional increase in ROS by red/NIR light would

provide neuroprotective eﬀects. From the current literature, it is clear

that the mechanism for the eﬀect of red/NIR light on ROS production,

in addition to the cellular mechanisms responsible for balancing the

ROS levels required for maintaining optimal mitochondrial and cellular

function are not fully understood. However, since maintaining redox

homeostasis is paramount for the optimal functioning of the cell and

neuronal survival, we suggest that the exposure to red/NIR light may

restore redox homeostasis in pathological conditions where it is

perturbed. Therefore, in cells with elevated ROS levels, red/NIR light

may cause mild cellular stress by an unknown mechanism that may

induce an adaptive response, which includes the upregulation of genes

with a role in redox homeostasis. We suggest that the observed increase

in ·NO upon red/NIR light irradiation, by the various mechanisms

discussed in Section 5, may facilitate the production of peroxynitrite

K. Beirne et al. Mitochondrion 36 (2017) 85–95

through the reaction of ·NO with ROS. The elevated RNS levels may

induce an adaptive response by triggering a diﬀerent signaling pathway

to that which is triggered by ROS.

6.2. ROS/RNS as signaling intermediates

The upregulation of ROS and RNS, such as peroxynitrite, are

thought to promote cell viability and increase proliferation owing to

their ability to function as signal intermediates (Fig. 3). ROS, which is

produced by the ETC complexes as a byproduct of cellular respiration

permits communication from the mitochondria to the rest of the cell.

This mitochondrial signal transduction can activate various signaling

pathways resulting in the expression of a plethora of genes including

those involved either directly or indirectly in the suppression of

apoptosis, cell survival or cell proliferation. Curiously, among the genes

aﬀected by red/NIR light irradiation, were those with roles in anti-

oxidation (Tafur and Mills, 2008).

6.3. The mitochondrial signaling pathway

ROS is thought to mediate its protective eﬀect via the activation of

the redox sensitive transcription factor nuclear factor (NF)-ĸB which is

proposed to be sensitive to an increase in ROS generation. In support of

this theory it was found that an increase in ROS production by

mitochondrial inhibitors brought about a concurrent increase in

NFĸB, while the exposure to antioxidants reduced the NFĸB activation

(Chen et al., 2011). NFĸB can both induce and repress gene expression

by binding to ĸB elements in the promoter and enhancer regions of the

gene. This transcription factor has been shown to induce the expression

of numerous genes with functions in cell survival, the stress response

and inﬂammation (Chen et al., 2011). When superoxides are produced

in the mitochondria they are metabolized to H

which is thought to

activate NF-ĸB by triggering the dissociation of inhibitor of ĸB kinase

from NFĸB. This allows NF-ĸB to translocate to the nucleus where it can

alter gene expression (Fig. 3)(Gloire et al., 2006).

The redox sensitive signaling pathways however diﬀer with cell

type making it diﬃcult to predict the outcome of red/NIR light therapy

if attempting to ﬁnd a general cellular mechanism (Gloire et al., 2006).

It is no surprise that the results obtained appear to be inconsistent with

varying cell type. This may provide some explanation for the diﬀer-

ential results that have been observed with the use of red/NIR light

therapy in diﬀerent cell types. For example, irradiation of traumatized

muscle tissue with red light therapy reduced the levels of ROS and NFĸB

activation and the associated increase in the expression of proinﬂam-

matory genes (Rizzi et al., 2006). On the other hand NIR light has been

shown to induce ROS production and NFĸB activation in murine

embryonic ﬁbroblasts (Chen et al., 2011).

In a similar fashion, peroxynitrite production triggers this stress

response, causing the up or downregulation of cell signaling cascades in

a cell dependent manner (Liaudet et al., 2009). Peroxynitrite signaling

is mediated by tyrosine nitration of proteins, particularly those involved

in phosphotyrosine-dependent signaling. The oxidant is also involved in

signaling via mitogen activated protein kinase, protein kinase B and C,

NFĸB and the insulin receptor. Of note, these pathways which converge

on the upregulation of mediators of cell survival, growth and prolifera-

tion are also activated by other stress stimuli such as ROS.

In support of the theory that red/NIR light causes upregulation in

the expression of genes associated with cell survival, is the increased

neuroprotective eﬀects observed when red/NIR light is administered

before the induced injury in models of neurodegeneration, compared to

when administered after injury (Ying et al., 2008). This suggests that

red/NIR light could be mediating its protective eﬀects by instigating the

production of ROS/RNS at levels suﬃcient to cause the upregulation of

stress response genes, with negligible damage to the irradiated tissue. If

the same tissue was subsequently exposed to a toxic agent, the cell

would be equipped with the appropriate defense mechanisms to cope

with such an insult. The destructive eﬀects to the tissue in such an

instance would be remarkably less, such as is seen in various pretreat-

ment models (Albarracin et al., 2013; Rutar et al., 2012; Albarracin and

Valter, 2012). This knowledge could be exploited in neurodegenerative

diseases with a pre-symptomatic phase. The observation demonstrates

that red/NIR light therapy would be most eﬀective as a preventative

therapeutic treatment, administered before the onset of clinical symp-

toms and irreversible damage. With respect to retinal degenerative

disease this preventative treatment approach would be particularly

relevant in inherited optic neuropathies such as Leber's hereditary optic

Fig. 3. The upregulation of ROS or RNS by red/NIR light triggers the translocation of the transcription factor NFĸB to the nucleus, enabling NFĸB to alter gene expression.

K. Beirne et al. Mitochondrion 36 (2017) 85–95

neuropathy. Individuals with a LHON-causing mtDNA pathogenic

variant could be monitored so that treatment with red/NIR light could

begin upon detection of pre-symptomatic abnormalities as a pre-

treatment for the clinical symptoms. In the event of a more acute

neurodegenerative condition such as TBI, exploiting the enhanced

therapeutic eﬀects of red/NIR light observed when used as a pretreat-

ment would not be feasible. Therefore, the neuroprotective potential of

red/NIR light therapy, as seen in the pre-treated animal models, may be

somewhat limited in a real life clinical setting.

7. The eﬀects of red/NIR light on the anti-inﬂammatory response

Peroxynitrite and ROS are also produced by macrophages during

inﬂammation, which are responsible for the cytotoxic eﬀects (Kang

et al., 2002). As mentioned above, red/NIR light has shown the ability

to reduce the presence of these reactive species in irradiated tissues,

thereby attenuating some of the cytotoxic eﬀects of the inﬂammatory

response. Furthermore, modulation of the immune response, itself, was

seen in many studies with red/NIR light treatment (Begum et al., 2013;

Kokkinopoulos et al., 2013; Gavish et al., 2008). Yet, how red/NIR light

mediates this anti-inﬂammatory eﬀect is poorly understood.

Mitochondria play a major role in the activation of the inﬂamma-

some, a molecular platform that activates proapoptotic proteins,

cytokines and other mediators of inﬂammation upon detection of

infectious agents or cellular damage (Green et al., 2011). ROS can

similarly activate the inﬂammasome. The production of mitochond-

rially-derived ROS is known to increase when mitochondria are

dysfunctional. Decreased electron ﬂux through the electron transport

chain reduces the ability of COX to fully reduce oxygen to water,

thereby increasing the formation of ROS. The action of red/NIR light in

improving the functions of the mitochondrial electron transport chain

could reduce the amount of ROS generated, which may reduce the

activation of the inﬂammasome. Alternatively, the mitochondrial signal

transduction triggered by an increase in ROS/RNS as discussed above,

could also upregulate the expression of anti-inﬂammatory or antiox-

idants proteins, to either directly or indirectly dampen the inﬂamma-

tory response. However, it has been found that low level laser

irradiation (LLLI) can reduce the gene expression of anti-inﬂammatory

cytokines as well as pro-inﬂammatory cytokines (Gavish et al., 2008).

In this particular situation LPS was used to elicit an inﬂammatory like

phenotype, triggering the expression of pro-inﬂammatory cytokines in

addition to the anti-inﬂammatory cytokine, IL-10. The anti-inﬂamma-

tory cytokine is also triggered during the inﬂammatory response to

limit the host immune response, thereby minimising the damage caused

to the aﬀected tissue during the inﬂammatory response (Iyer and

Cheng, 2012). The ﬁndings demonstrate that LLLI is able to dampen

the entire inﬂammatory response as oppose to speciﬁcally down-

regulating proinﬂammatory cytokines, suggesting that red/NIR light

may be acting further upstream, inﬂuencing factors governing the

inﬂammatory response. Also, there appears to be a correlation between

the inﬂammatory level in the tissue and the extent of the inhibition of

the inﬂammatory response (Gavish et al., 2008). It was found that LLLI

had little eﬀect on the inﬂammatory response at low levels of

inﬂammation, but, produced a potent anti-inﬂammatory eﬀect at high

levels of inﬂammation (Gavish et al., 2008). This shows an ability of

red/NIR light to restore homeostasis in the tissue. In fact, ROS is

produced by the microglia itself, and plays an important role in the

induction of pro-inﬂammatory genes (Innamorato et al., 2009). There-

fore, restoring the redox balance in microglia may facilitate the

transition of the microglia from an activated to a resting state.

It has been suggested that the mitochondrial dysfunction seen in

many neurodegenerative diseases not only aﬀects neurons, but also the

microglia (Ferger et al., 2010). Experimental ﬁndings have shown that

complex I inhibition in microglia inhibited the IL-4 mediated reduction

in pro-inﬂammatory cytokines and the secretion of the neuroprotective

insulin-like growth factor-1. The inﬂammatory response triggered by

pathogens or damaged neurons functions to protect the neural tissue,

but if this response is not attenuated the response would switch from a

protective one to a deleterious one (Ramesh et al., 2013). When

activated, microglia secrete anti-inﬂammatory cytokines, in addition

to pro-inﬂammatory cytokines to control the inﬂammatory response,

preventing unnecessary damage to neural tissue. Electron transport

deﬁciencies in microglia, appear to perturb the anti-inﬂammatory arm

of the immune response (Ferger et al., 2010). Since red/NIR light has

been shown to improve the function of the electron transport chain in

neurons, it can be suggested that a similar eﬀect would be seen in

microglia with mitochondrial dysfunction.

8. Conclusion

Wavelengths of light in the red to NIR region of the electromagnetic

spectrum are optimal for the photostimulation of mitochondria as a

treatment for neurodegeneration in the retina. The photon energy of

red/NIR light appears to correspond to the absorption maxima of

chromophores present in complex IV of the electron transport chain,

triggering biostimulatory eﬀects. Additionally, red and NIR light have

an increased ability to penetrate tissue and are associated with

signiﬁcantly less safety concerns than light of shorter wavelengths.

The potential for this treatment to be a success in patients is high. This

is supported, ﬁrstly, by the potent neuroprotective eﬀects demonstrated

in various models of retinal damage. Secondly, data from human

studies, albeit limited, shows the ability of red and NIR light to improve

visual function. Furthermore, no adverse eﬀects were observed in these

previously published studies. The ﬁndings suggest that red/NIR light

therapy is safe and eﬀective as a non-invasive treatment for retinal

neurodegeneration.

Much evidence has been gathered in eﬀorts to elucidate the under-

lying mechanism responsible for the neuroprotective eﬀects of red/NIR

light, but the mechanism remains unclear. Experimental ﬁndings

suggest that there are many possible molecular and cellular eﬀects of

red/NIR light, which could all contribute to the observed neuroprotec-

tive eﬀects, when explored separately, but when taken collectively

some eﬀects appear to contradict others. Improved neuronal mitochon-

drial function, increased blood ﬂow to neural tissue, an increase in ·NO

levels, slowing down of the reactivation of the mitochondrial electron

transport chain during reperfusion, increased mitochondrial biogenesis,

reduction in ROS levels, a reduction in ·NO levels, upregulation of cell

survival mediators and restoration of normal microglial function have

all been proposed as potential underlying mechanisms of red/NIR light.

The increased ability of the neuron to survive during challenging

conditions may be due to the resulting net eﬀect of a number of red/

NIR light induced molecular and cellular events. The eﬀects that prevail

in a particular cell/tissue may depend on the state of the cell/tissue at

the time of irradiation and the subsequent challenges to which the cell

is exposed to.

Aside from the fact that the mechanism of action is unclear, the lack

of consensus surrounding the optimal parameters of red/NIR light, such

as irradiance, radiant exposure and wavelength, for diﬀerent conditions

is a cause for concern. Although beneﬁcial eﬀects have been found

using several diﬀerent parameter combinations, eﬀorts to ﬁnd the best

possible eﬀect with the least possible risk are, for the most part, not

done. This unconventional experimental approach has been established

based on the assumption that red/NIR light therapy is safe, but this

approach would not be tolerated for any other therapeutic interven-

tions. In order for advances to be made in this ﬁeld a much more

detailed collection of experiments needs to be done to establish the

optimal parameters for each condition with a potential for treatment

with red/NIR light.

Acknowledgements

This research was funded by Fight for Sight UK (505025).

K. Beirne et al. Mitochondrion 36 (2017) 85–95

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670nm photobiomodulation modulates bioenergetics and oxidative stress, in rat Müller cells challenged with high glucose

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Dec 2021
PLOS ONE

Diabetic retinopathy (DR), the most common complication of diabetes mellitus, is associated with oxidative stress, nuclear factor-κB (NFκB) activation, and excess production of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). Muller glial cells, spanning the entirety of the retina, are involved in DR inflammation. Mitigation of DR pathology currently occurs via invasive, frequently ineffective therapies which can cause adverse effects. The application of far-red to near-infrared (NIR) light (630-1000nm) reduces oxidative stress and inflammation in vitro and in vivo . Thus, we hypothesize that 670nm light treatment will diminish oxidative stress preventing downstream inflammatory mechanisms associated with DR initiated by Muller cells. In this study, we used an in vitro model system of rat Müller glial cells grown under normal (5 mM) or high (25 mM) glucose conditions and treated with a 670 nm light emitting diode array (LED) (4.5 J/cm ² ) or no light (sham) daily. We report that a single 670 nm light treatment diminished reactive oxygen species (ROS) production and preserved mitochondrial integrity in this in vitro model of early DR. Furthermore, treatment for 3 days in culture reduced NFκB activity to levels observed in normal glucose and prevented the subsequent increase in ICAM-1. The ability of 670nm light treatment to prevent early molecular changes in this in vitro high glucose model system suggests light treatment could mitigate early deleterious effects modulating inflammatory signaling and diminishing oxidative stress.

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Preprint

Full-text available

Jan 2021

Diabetic retinopathy (DR), the most common complication of diabetes mellitus, is associated with oxidative stress, nuclear factor-kB (NFkB) activation, and excess production of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). Current therapies are invasive, frequently ineffective, and have adverse effects. The application of far-red to near-infrared (NIR) light (630-1000nm) reduces oxidative stress and inflammation in vitro and in vivo . Thus, we hypothesize that 670nm light treatment will dimish oxidative stress preventing downstream inflammatory mechanisms associated with DR. We used an in vitro model system of rat Müller glial cells grown under normal (5 mM) or high (25 mM) glucose conditions and treated with a 670 nm light emitting diode array (LED) (4.5 J/cm ² ) or no light (sham) daily. We report that a single 670 nm light treatment diminished ROS production and preserved mitochondrial integrity and ATP production in this in vitro model of diabetic retinopathy. Furthermore, treatment for 3 days in culture reduced NFkB activity to levels observed in normal glucose and prevented the subsequent increase in ICAM-1. The ability of 670nm light treatment to prevent early molecular changes in this established DR model system suggests light treatment could become an early therapeutic option for DR.

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Aug 2021
INT J MOL SCI

Retinal ganglion cells (RGCs) undergo dendritic pruning in a variety of neurodegenerative diseases, including glaucoma and autosomal dominant optic atrophy (ADOA). Axotomising RGCs by severing the optic nerve generates an acute model of RGC dendropathy, which can be utilized to assess the therapeutic potential of treatments for RGC degeneration. Photobiomodulation (PBM) with red light provided neuroprotection to RGCs when administered ex vivo to wild-type retinal explants. In the current study, we used aged (13–15-month-old) wild-type and heterozygous B6;C3-Opa1Q285STOP (Opa1+/−) mice, a model of ADOA exhibiting RGC dendropathy. These mice were pre-treated with 4 J/cm2 of 670 nm light for five consecutive days before the eyes were enucleated and the retinas flat-mounted into explant cultures for 0-, 8- or 16-h ex vivo. RGCs were imaged by confocal microscopy, and their dendritic architecture was quantified by Sholl analysis. In vivo 670 nm light pretreatment inhibited the RGC dendropathy observed in untreated wild-type retinas over 16 h ex vivo and inhibited dendropathy in ON-center RGCs in wild-type but not Opa1+/− retinas. Immunohistochemistry revealed that aged Opa1+/− RGCs exhibited increased nitrosative damage alongside significantly lower activation of NF-κB and upregulation of DJ-1. PBM restored NF-κB activation in Opa1+/− RGCs and enhanced DJ-1 expression in both genotypes, indicating a potential molecular mechanism priming the retina to resist future oxidative insult. These data support the potential of PBM as a treatment for diseases involving RGC degeneration.

Near Infrared (NIR) Light Therapy of Eye Diseases: A Review

Article

Full-text available

Jan 2021
Int J Med Sci

Near infrared (NIR) light therapy, or photobiomodulation therapy (PBMT), has gained persistent worldwide attention in recent years as a new novel scientific approach for therapeutic applications in ophthalmology. This ongoing therapeutic adoption of NIR therapy is largely propelled by significant advances in the fields of photobiology and bioenergetics, such as the discovery of photoneuromodulation by cytochrome c oxidase and the elucidation of therapeutic biochemical processes. Upon transcranial delivery, NIR light has been shown to significantly increase cytochrome oxidase and superoxide dismutase activities which suggests its role in inducing metabolic and antioxidant beneficial effects. Furthermore, NIR light may also boost cerebral blood flow and cognitive functions in humans without adverse effects. In this review, we highlight the value of NIR therapy as a novel paradigm for treatment of visual and neurological conditions, and provide scientific evidence to support the use of NIR therapy with emphasis on molecular and cellular mechanisms in eye diseases.

Emerging model systems and treatment approaches for Leber's hereditary optic neuropathy: Challenges and opportunities

Article

Feb 2020
BBA-MOL BASIS DIS

Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease mainly affecting retinal ganglion cells (RGCs). The pathogenesis of LHON remains ill-characterized due to a historic lack of effective disease models. Promising models have recently begun to emerge; however, less effective models remain popular. Many such models represent LHON using non-neuronal cells or assume that mutant mtDNA alone is sufficient to model the disease. This is problematic because context-specific factors play a significant role in LHON pathogenesis, as the mtDNA mutation itself is necessary but not sufficient to cause LHON. Effective models of LHON should be capable of demonstrating processes that distinguish healthy carrier cells from diseased cells. In light of these considerations, we review the pathophysiology of LHON as it relates to old, new and future models. We further discuss treatments for LHON and unanswered questions that might be explored using these new model systems.

Effect of Low-Level Laser on Some Metals Related to Redox State and Histological Alterations in the Liver and Kidney of Irradiated Rats

Article

Full-text available

Apr 2020
BIOL TRACE ELEM RES

Low-level laser therapy (LLLT) is a type of medicine that uses laser light at low levels to activate the cellular chromophores and the initiation of cellular signaling. This study aimed to evaluate the photomodulation effect of LLL against ionizing radiation (IR)–induced metal disorders related to redox state in the liver and kidney of male rats. Rats were divided into 4 groups (control, LLLT, IR (7Gy), IR+LLLT). The results showed that LLLT 870 nm one time for 3 days post-irradiation revealed redistribution of iron (Fe), copper (Cu), zinc (Zn),calcium (Ca), magnesium (Mg), manganese (Mn), and selenium (Se) in the liver and kidney tissues. Moreover, LLLT attenuated the oxidative stress manifested by a marked reduction of hydrogen peroxide (H2O2), 4-hydroxynonenal (4-HNE), total oxidant state (TOS), and oxidative stress index (OSI) associated with a significant increase in total antioxidant status (TAS), glutathione (GSH) content, and glutathione peroxide (GPx), glutathione reductase (GRx), superoxide dismutase(SOD), and catalase (CAT) activities. Moreover, LLLT displayed an increase in glutathione-S-transferase (GSH-T) and ceruloplasmin activities and a decrease in the activity of gamma-glutamyl transferase (γ-GT). Besides, LLLT significantly attenuated the histological changes in the liver and kidney tissues, denoted by a reduction in the necrotic and degenerative changes of hepatocytes and an improvement in the corpuscles and tubules of the kidney. In conclusion, LLLT could be used as an adjuvant treatment post-exposure to radiation, while it is not beneficial to use it on the normal tissue.

Chronic photodamage in the chicken retina using 650-nm semiconductor laser

Article

Full-text available

Jun 2019

Aim: To investigate the occurrence of chronic photodamage in the cone-based retina, following long-term exposure to a 650-nm semiconductor laser (power: 2 mW). Methods: Chickens fed for 1mo under natural light after hatching were irradiated with 650-nm laser light at different times each day. Fifteen animals were included in each group. Group A was a control group, irradiated with natural light during the entire study. Group B was irradiated with laser for 3 min/d. Group C was irradiated with laser for 6 min/d. Group D was irradiated with laser for 30 min/d. The duration of the light experiment was 6mo. We obtained data at 1, 3, and 6mo, including measuring the retinal thickness in vivo using optical coherence tomography, hematoxylin and eosin staining, TUNEL assay, apoptosis staining, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and Western blotting to detect changes in L/M opsins and rhodopsin. Results: At 1mo, the MDA content in Group D was higher than that observed in Group A (P=0.019). At 3mo the MDA content in Groups C and D was higher than that reported in Group A (P=0.026, 0.003). At 6mo, the MDA content in Groups B, C, and D was higher than that observed in Group A (P=0.038, 0.032, 0.000, respectively). There was no difference in SOD activity, and L/M opsin and rhodopsin content between the groups at 1 and 3mo. The SOD activity in group D was significantly decreased at 6mo (P=0.000), as was the content of rhodopsin. There was no significant reduction observed in retinal thickness, abnormal cell arrangement, and positive staining of TUNEL in the groups during the 6-month study period. Conclusion: Irradiation using a 650 nm semiconductor laser (power: 2 mW) for 6min per day over 6mo do not cause photodamage. Similarly, a 3-month exposure of 30min per day do not cause damage. However, irradiation for 6mo resulted in a significant increase in the content of free radicals and a decrease in the content of rhodopsin in the retina, suggesting the presence of photodamage.

Photoactivation of cytochrome с oxidase activity in liver mitochondria of Japanese quail by therapeutic doses of blue and red LED irradiation

Article

Sep 2018

Цель исследования - выяснение влияния синего (450 нм) и красного (630 нм) светодиодного облучения различной энергетической экспозиции на активность митохондриальной цитохром с-оксидазы. Методики. Объектом исследования служили митохондрии, изолированные из печени японского перепела Coturnix japonica . Цитохром с-оксидазную активность измеряли по скорости окисления восстановленного тетраметил-р-фенилендиамина в присутствии ротенона. Результаты. Показано, что облучение митохондрий синим светом с энергетическими экспозициями, не превышающими 4 Дж/см вызывает примерно 5-15-процентную стимуляцию активности цитохром с-оксидазы, а дозы больше 5 Дж/см приводят к ингибированию активности фермента. Облучение митохондрий красным светом также оказывает стимулирующее действие (10-20%) на цитохром с-оксидазную активность по сравнению с необлученными образцами. Заключение. Предполагается, что низкодозовое облучение синим светом может иметь сходное с красным светом терапевтическое действие при фотобиомодуляции. The aim was to study the effect of blue (450 nm) and red (630 nm) LED irradiation with different energy exposures on activity of mitochondrial cytochrome c oxidase. Methods. The study was performed on mitochondria isolated from the liver of Japanese quail Coturnix japonica . Cytochrome c oxidase activity was measured by the rate of oxidation of reduced tetramethyl-p-phenylenediamine in the presence of rotenone. Results. Irradiation of mitochondria with blue light at doses not exceeding 4 J/cm caused approximately 5-15% stimulation of cytochrome c oxidase activity while doses higher than 5 J/cm led to inhibition of this enzyme. Irradiation of mitochondria with red light also exerted a slight stimulating effect (10-20%) on cytochrome c oxidase activity compared to unirradiated samples. Conclusion. The study suggested that low-dose irradiation with blue light may produce a therapeutic effect similar to red light in photobiomodulation.

Transcranial, Red/Near-Infrared Light-Emitting Diode Therapy to Improve Cognition in Chronic Traumatic Brain Injury

Article

Full-text available

Dec 2016

Objective: We review the general topic of traumatic brain injury (TBI) and our research utilizing transcranial photobiomodulation (tPBM) to improve cognition in chronic TBI using red/near-infrared (NIR) light-emitting diodes (LEDs) to deliver light to the head. tPBM improves mitochondrial function increasing oxygen consumption, production of adenosine triphosphate (ATP), and improving cellular energy stores. Nitric oxide is released from the cells increasing regional blood flow in the brain. Review of published studies: In our previously published study, 11 chronic TBI patients with closed-head TBI caused by different accidents (motor vehicle accident, sports-related, improvised explosive device blast injury) and exhibiting long-lasting cognitive dysfunction received 18 outpatient treatments (Monday, Wednesday, Friday for 6 weeks) starting at 10 months to 8 years post-TBI. LED therapy is nonthermal, painless, and noninvasive. An LED-based device classified as nonsignificant risk (FDA cleared) was used. Each LED cluster head (5.35 cm diameter, 500mW, 22.2 mW/cm2) was applied for 9 min 45 sec (13 J/cm2) using 11 locations on the scalp: midline from front-to-back hairline and bilaterally on frontal, parietal, and temporal areas. Testing was performed before and after transcranial LED (tLED; at 1 week, 1 month, and at 2 months after the 18th treatment) and showed significant improvements in executive function and verbal memory. There were also fewer post-traumatic stress disorder (PTSD) symptoms reported. Ongoing studies: Ongoing, current studies involve TBI patients who have been treated with tLED using either 26 J/cm2 per LED location on the head or treated with intranasal only (iLED) using red (633 nm) and NIR (810 nm) diodes placed into the nostrils. The NIR iLED is hypothesized to deliver photons to the hippocampus, and the red 633 nm iLED is believed to increase melatonin. Results have been similar to the previously published tLED study. Actigraphy sleep data showed increased time asleep (on average one additional hour per night) after the 18th tLED or iLED treatment. LED treatments may be performed in the home. Sham-controlled studies with veterans who have cognitive dysfunction from Gulf War Illness, blast TBI, and TBI/PTSD are currently ongoing.

Transcranial Low-Level Laser (Light) Therapy for Brain Injury

Article

Full-text available

Dec 2016

Background: Low-level laser therapy (LLLT) or photobiomodulation (PBM) is a possible treatment for brain injury, including traumatic brain injury (TBI). Methods: We review the fundamental mechanisms at the cellular and molecular level and the effects on the brain are discussed. There are several contributing processes that have been proposed to lead to the beneficial effects of PBM in treating TBI such as stimulation of neurogenesis, a decrease in inflammation, and neuroprotection. Both animal and clinical trials for ischemic stroke are outlined. A number of articles have shown how transcranial LLLT (tLLLT) is effective at increasing memory, learning, and the overall neurological performance in rodent models with TBI. Results: Our laboratory has conducted three different studies on the effects of tLLLT on mice with TBI. The first studied pulsed against continuous laser irradiation, finding that 10 Hz pulsed was the best. The second compared four different wavelengths, discovering only 660 and 810 nm to have any effectiveness, whereas 732 and 980 nm did not. The third looked at varying regimens of daily laser treatments (1, 3, and 14 days) and found that 14 laser applications was excessive. We also review several studies of the effects of tLLLT on neuroprogenitor cells, brain-derived neurotrophic factor and synaptogenesis, immediate early response knockout mice, and tLLLT in combination therapy with metabolic inhibitors. Conclusions: Finally, some clinical studies in TBI patients are covered.

Photobiomodulation reduces drusen volume and improves visual acuity and contrast sensitivity in dry age-related macular degeneration

Article

Full-text available

Dec 2016

Purpose: To evaluate the efficacy of photobiomodulation (PBM) treatment for patients with dry age-related macular degeneration (AMD). Methods: Assessments on 42 eyes with dry AMD (age related eye disease study (AREDS) 2-4) were conducted. Multiwavelength light emitting diode (LED) light comprising of yellow (590 nm), red (670 nm) and near-infrared (790 nm) bandwidths was applied to subjects' eyes for a treatment course of 3 weeks. Outcome measures were changes in best-corrected visual acuity (BCVA), contrast sensitivity (CS), drusen volume and central drusen thickness. Results: Significant improvement in mean BCVA of 5.90 letters (p < 0.001) was seen on completion of the 3-week treatment and 5.14 letters (p < 0.001) after 3 months. Contrast sensitivity improved significantly (log unit improvement of 0.11 (p = 0.02) at 3 weeks and 3 months (log unit improvement of 0.16 (p = 0.02) at three cycles per degree. Drusen volume decreased by 0.024 mm(3) (p < 0.001) and central drusen thickness was significantly reduced by a mean of 3.78 μm (p < 0.001), while overall central retinal thickness and retinal volume remained stable. Conclusion: This is the first study demonstrating improvements in functional and anatomical outcomes in dry AMD subjects with PBM therapy. These findings corroborate an earlier pilot study that looked at functional outcome measures. The addition of anatomical evidence contributes to the basis for further development of a non-invasive PBM treatment for dry AMD.

Treatment of dry age-related macular degeneration with photobiomodulation

Article

Full-text available

Jan 2012

Objective: To evaluate if LED Photobiomodulation (PBM) can affect vision in patients with dry Age- Related Macular Degeneration (AMD). Methods: Prospective interventional case series. Near Infra Red (NIR) and yellow wavelengths of low powered LED light were applied to eyes with AMD in serial consecutive treatments. Included were patients with dry AMD, 50 years or older and with visual acuity between 20/20-20/200. Primary outcome measures selected were change in visual acuity, contrast sensitivity and fixation stability. Results: The treatment protocol was completed in 18 eyes (9 patients). Changes in visual acuity (p<0.0001) and contrast sensitivity (p<0.0001 at 3 cycles/degree and p<0.0032 at 1.5 cycles/degree) were positive and significant. There were no significant changes in fixation stability parameters. Conclusions: LED PBM proves to be beneficial for improvement of vision and contrast sensitivity as well as a safe treatment for dry AMD in this pilot study. Larger studies are warranted to validate the findings from this study.

Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain

Article

Full-text available

Jan 2013
MEDIAT INFLAMM

Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in an AAV-Based Rat Genetic Model of Parkinson's Disease

Article

Full-text available

Oct 2015
PLOS ONE

Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson's disease (PD). In this model, daily exposure of both sides of the rat's head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies.

“Transcranial Low-Level Laser (Light) Therapy for Stroke and Traumatic Brain Injury in Animal Models.”

Chapter

Aug 2016

Low-level laser (light) therapy (LLLT) for traumatic events that cause brain damage is currently an experimental concept. The broad goals for clinical utilization are the prevention of brain damage, relief of symptoms, and stimulation of the repair process. Experimental studies have tested and continue to test these goals by investigating LLLT in animal models of stroke and traumatic brain injury (TBI). Animal studies of tLLLT for ischemic stroke have been summarized in this chapter. Studies by other groups on LLLT in mouse TBI models have been discussed. Four studies from our laboratory have been described. The ﬁrst study looked at pulsed versus continuous wave (CW) laser irradiation in a controlled cortical impact (CCI)-TBI model and found pulsed 810 nm laser at 10 Hz to be superior. The second study looked at four diﬀerent laser wavelengths (660, 730, 810, and 980 nm) in a closed-head TBI model; only 660 nm and 810 nm were eﬀective. The third study looked at diﬀerent treatment repetition regimens (1, 3, and 14 daily laser treatments). The last study used immunoﬂuorescence techniques to show that neurogenesis, brain-derived neurotrophic factor (BDNF), and synaptogenesis were upregulated after transcranial LLLT (tLLLT) for TBI. Limitations in knowledge are still apparent, such as the optimal wavelength, light source, doses, pulsed or CW, polarization state, treatment timing, and repetition frequency. Collaborative eﬀorts between clinicians and basic researchers will likely increase the usage and understanding of eﬀective laser-based therapies in the central nervous system (CNS).

Red Light Treatment in an Axotomy Model of Neurodegeneration

Article

Jun 2016

Red light has been shown to provide neuroprotective effects. Axotomising the optic nerve initiates retinal ganglion cell (RGC) degeneration, and an early marker of this is dendritic pruning. We hypothesised that 670 nm light can delay axotomy induced dendritic pruning in the retinal explant. To test this hypothesis, we monitored the effects of 670 nm light (radiant exposure of 31.7 J/cm(2) ), on RGC dendritic pruning in retinal explants from C57BL/6J mice, at 40 minutes, 8 hours and 16 hours post axotomy. For sham-treated retinae, area under the Sholl curve, peak of the Sholl curve and dendritic length at 8 hours post axotomy showed statistically significant reductions by 42.3% (p=0.008), 29.8% (p=0.007) and 38.4% (p=0.038), respectively, which were further reduced after 16 hours by 40.56% (p<0.008), 33.9% (p<0.007), 45.43% (p<0.006), respectively. Dendritic field area was also significantly reduced after 16 hours, by 44.23% (p<0.019). Such statistically significant reductions were not seen in light-treated RGCs at 8 or 16 hours post axotomy. The results demonstrate the ability of 670 nm light to partially prevent ex vivo dendropathy in the mouse retina, suggesting that it is worth exploring as a treatment option for dendropathy associated neurodegenerative diseases, including glaucoma and Alzheimer's disease. This article is protected by copyright. All rights reserved.

Repeated transcranial low-level laser therapy for traumatic brain injury in mice: biphasic dose response and long-term treatment outcome

Article

Mar 2016
J BIOPHOTONICS

We previously showed that near-infrared laser photobiomodulation (PBM) (810 nm, CW, 18 J/cm(2) , 25 mW/cm(2) ) delivered to the mouse daily for 3-days after a controlled cortical impact traumatic brain injury (TBI) gave a significant improvement in neurological/cognitive function. However the same parameters delivered 14X daily gave significantly less benefit. This biphasic dose response intrigued us, and we decided to follow the mice that received 3X or 14X laser treatments out to 56-days post-TBI. We found the 14X group showed worse neurological function than the no-treatment TBI group at 2-weeks, but started to improve steadily during the next 6-weeks, and by 56-days were significantly better than the no-treatment TBI mice, but still worse than the 3X mice. A marker of activated glial cells (GFAP) was significantly increased in the brain regions (compared to both untreated TBI and 3X groups) at 4-weeks in the 14X group, but the GFAP had fallen to low levels in both 3X and 14X groups by 8-weeks. We conclude that an excessive number of laser-treatments delivered to mice can temporarily inhibit the process of brain repair stimulated by tPBM, but then the inhibitory effect ceases, and brain repair can resume. The mechanism may be temporary induction of reactive gliosis.

Temperature profiles in human retina and choroid during laser coagulation with different wavelengths ranging from 514 to 810 nm

Article

Jan 1992

Temperature profiles in human retina and choroid during laser coagulation were calculated for five different laser wavelengths: 514, 580, 620, 647 and 810 nm. For this, the spectral absorption of melanin and hemoglobin, the distribution of these chromophores in retina and choroid in vivo, and heat conduction were taken into account. The coagulation parameters chosen were a spot size of 500 μm and an exposure time of 100 msec. All curves were normalized to the same temperature rise at the retinal pigment epithelium. It turned out that the temperature profiles in the retina and inner choroid were very similar for all wavelengths, because their shape depends mainly on heat conduction from the strongly absorbing RPE and from the choroid. In the mid and outer choroid, however, a much higher temperature rise was observed at long wavelengths, especially at 810 nm, than at 514 and 580 nm. Discussing the therapeutic consequences of these findings, the conclusion is drawn that green laser wavelengths are best suited for most clinical applications of photocoagulation.