Infrared (IR) light is ubiquitously present to most life on the earth. Of the total amount of solar energy reaching the human skin, 54% is IR and 30% is IR type A—near-infrared—(NIR; with a wavelength range of 760 to 1440 nm),1 which penetrates through the human skin and reaches deeply into tissue, depending on wavelength and energy.2
NIR is used to treat a variety of conditions such as muscle pain,3 wounds,4 neuropathic pain,5 and headache.6 NIR is also used for wellness and lifestyle purposes such as for cosmetic improvement in peri-orbital wrinkles.7,8 The clinical use of NIR light applied in NIR-spectroscopy dates from the mid-1980s, when it was used for monitoring of the brain in the neonate and the fetus.9
The use of transcranial phototherapy for treating brain disorders started with its application to acute stroke. Numerous preclinical animal studies1011.–12 suggested that the application of NIR laser (810 nm) to the head at various times (hours) after induction of an acute stroke had beneficial effects on subsequent neurological performance and reduced lesion size. Evidence was obtained for the anti-inflammatory, anti-apoptotic, and proneurogenesis effects in the brain stimulated by this approach.13,14 These promising animal studies led to the conduction of a series of clinical trials called NeuroThera Effectiveness and Safety Trials (NEST). All together there were three large studies conducted in 1410 stroke patients [NEST-1 (
), NEST-2 (
)] that demonstrated that NIR light delivered transcranially with a class-IV laser is safe, with no significant differences in rates of adverse events with NIR, when compared to sham exposure.1516.–17 Other preclinical studies and clinical trials have suggested that transcranial photobiomodulation (PBM: laser or light emitting diodes—LED) is safe and effective for acute1819.20.21.–22 and chronic2324.–25 traumatic brain injury (TBI) and has beneficial effects on neurodegenerative diseases (Alzheimer’s and Parkinson’s).26,27
For the transcranial treatment of major depressive disorder (MDD), both PBM LEDs and lasers have been experimentally tested, although PBM is not FDA-approved for the treatment of MDD. Certain forms of PBM treatment are also referred to as low-level light therapy (LLLT), since it utilizes light at a low power (0.1 to 0.5 W output at the source) to avoid any heating of tissue. The irradiance of the PBM medical devices (or power density) typically ranges from 1 to 10 times the NIR irradiance from sunlight on the skin (
at the zenith). However, most PBM medical devices only deliver light energy at one or two selected wavelengths, as opposed to the whole spectrum of IR that is contained in sunlight. To our knowledge and to this date, transcranial PBM treatment has not caused any retinal injury—one of the most likely postulated adverse events, although care is taken routinely in such studies to protect the eyes with goggles or eye covers.28
In this review, we will first discuss the mechanisms of action by which NIR and red light (PBM) might improve symptoms of depression, and then present the clinical evidence for their use as a treatment for MDD and other comorbid psychiatric syndromes.
We found clinical and preclinical studies via PubMed search (December 15, 2015), using the following keywords: “near-infrared radiation,” “NIR,” “low-level light therapy,” “low-level laser therapy,” or “LLLT” plus “depression.” We chose studies that had a clinical focus, and we excluded studies involving NIR spectroscopy. We also located studies using the references from the articles found in the PubMed search. As the searched literature encompassed different conditions and disorders frequently comorbid with depression, a specific section of this review was devoted to the effect of PBM on psychiatric comorbidity. In the latter section, the following conditions were included, based on available literature: TBI, anxiety and post-traumatic stress syndromes, insomnia, and suicidal ideation. The literature search for the use of PBM to treat comorbid conditions was neither systematic nor extensive, but rather a secondary focus of this review. The information is presented in an organized fashion to allow the reader to easily grasp the potential applications of PBM for the treatment of depression and of its comorbid conditions. To attain this goal, the authors have allowed a margin of redundancy, by distributing different information derived from any given publication in separate sections of this review. To avoid an artificial inflation of the extant literature on the chosen topic, we referenced the main authors—and when appropriate their affiliation—when referring to the same articles more than once. The reader will find a table summarizing the six key clinical articles reviewed, also to avoid unintended inflation of the literature. The six clinical reports included in this review where extracted from a pool of 58 articles, that were originally identified with the literature search.
In addition, we used PubMed to find articles that examined the link between PBM and each of the various biological processes including metabolism, inflammation, oxidative stress, and neurogenesis.
Multiple studies have reported regional and global hypometabolism in MDD, which could be related (either causally or consequentially) to the neurobiology of mood disorders.2930.31.–32 Positron emission tomography studies have shown abnormalities in glucose consumption rates and in blood flow in several brain regions of subjects with major depression.33 Moreover, metabolic abnormalities in the anterior cingulate, the amygdala-hippocampus complex, the dorsolateral prefrontal cortex (DLPFC), and inferior parietal cortex seem to improve after antidepressant treatment or after recovery.3435.–36
With phosphorus magnetic resonance spectroscopy (
), the baseline pool of nucleotide triphosphate (NTP)—a product of the cellular utilization of glucose and a marker of the cellular energy availability—was low in subjects who subsequently responded to antidepressant treatment.32 Iosifescu et al.32 also demonstrated for the first time with
metabolite changes were noted in brain-only voxels of responders, but not in nonresponders to antidepressants.
In experimental and animal models, PBM (NIR and red light) noninvasively delivers energy to the cytochrome c oxidase and by stimulating the mitochondrial respiratory chain leads to increased ATP production (see Fig. 1).3738.–39 A study of the effects of NIR on patients with MDD found that a single session of NIR led to a marginally significant increase in regional cerebral blood flow.40 Whether the observed changes in cerebral blood flow resulted from fundamental changes in neuronal metabolism or changes in vascular tone remain to be clarified. Given the correlation of both hypometabolism and abnormal cerebral blood flow with MDD, the beneficial effect of NIR on brain metabolism is one potential mechanism for its antidepressant effect.
Animal and clinical research suggests that the inflammatory arm of the immune system contributes to MDD. Post-mortem gene expression profiling on tissue samples from Brodmann area 10 (BA10—prefrontal cortex) have shown that MDD is characterized by increased inflammation and apoptosis.41 In a case-control study, Simon et al.42 found that antidepressant-naive MDD subjects had significant elevations in the following cytokines and chemokines when compared to healthy controls:
. Although IL-10 is an anti-inflammatory cytokine, the results suggested that the elevated levels of this IL-10 were likely induced in response to the overall elevation of proinflammatory cytokine levels. In a review of the research on inflammation in MDD, Raison et al.43 proposed that proinflammatory cytokines might cause brain abnormalities that are characteristic of MDD. Indeed, animal research has shown that IL-1 mediates chronic depression in mice by suppressing hippocampal neurogenesis.44
One proinflammatory cytokine that may be of particular relevance to depression is CSF IL-6 (IL6 measured in cerebrospinal fluid). In a recent report, patients with MDD had significantly higher CSF IL-6 levels compared to healthy controls; CSF IL-6 levels were significantly higher than in the serum, and there was no significant correlation between CSF and serum IL-6 levels.45 These findings are consistent with a prior report showing a positive correlation between CSF IL-6 levels and the severity of depression and suicide attempts, with the strongest correlation found in violent suicide attempters.46 One report in a smaller sample of depressed patients has shown that CSF IL-647 was lower or comparable to healthy controls.
NIR light and red light (600 to 1600 nm) decreased synovial IL-6 gene expression (decreased mRNA levels) in a rat model of rheumatoid arthritis.48 In another study, NIR (810 nm) used as a treatment for pain in patients with rheumatoid arthritis decreased production of the following proinflammatory cytokines:
, and IL-8.49 Khuman et al.50 showed that transcranial NIR improved cognitive function and reduced neuroinflammation as measured by Iba1+ activated microglia in brain sections from mice that had suffered a TBI. Finally, NIR (970 nm) has been found to be an effective treatment for inflammatory-type acne.51 In summary, it is reasonable to predict that transcranial NIR treatment would likewise have an anti-inflammatory effect in patients suffering from MDD.
Research has demonstrated a correlation between MDD and vulnerability to oxidative stress.52 For example, depression-induced rats show a significant decrease in glutathione peroxidase (GSH-Px) activity in the cortex.53 Glutathione (GSH) is the most abundant and one of the important antioxidants in the brain; GSH-Px enzymes protect against oxidative stress via reducing hydroperoxides and scavenging free radicals.54 GSH also appears reduced in the brains of MDD subjects.55 Additionally, a study by Sarandol et al.52 demonstrated that MDD patients have higher levels of malondialdehyde, a toxic molecule and a biomarker of oxidative stress.56 Moreover, depressed patients have more red blood cell (RBC) oxidation compared to healthy controls.52 In the same study, the authors found a significant positive correlation between RBC superoxide dismutase (SOD) activity and depression severity. SOD serves to catalyze the removal of the toxic superoxide radical.57 Thus, elevated SOD activity in depressed patients might indicate higher levels of oxidative stress. Finally, catalase activity and nitric oxide (NO) levels have also been shown to be lower in depressed patients than in healthy controls.58 Catalase is an enzyme that protects cells against damaging reactive oxygen species (ROS) via degradation of hydrogen peroxide to water and oxygen.59 NO has protective effects against cell damage, which are likely due to its pleiotropic functions in regulating antioxidant enzymes and many other aspects of cell metabolism.60,61
Oxidative stress may be an effective target for antidepressant treatments. However, successful treatments for MDD vary in regard to their protective effects against oxidative stress.52,53,62 Animal research suggests that PBM may have beneficial effects on oxidative stress. In a rat model of traumatized muscle, NIR (904 nm) blocked the release of harmful ROS and the activation of the transcription factor, nuclear factor κB (NF-κB), both induced by muscle trauma. Trauma activates NF-κB by destroying a specific protein inhibitor of NF-κB called IκB, and this destruction was inhibited by NIR light. Furthermore, NIR reduced the associated overexpression of the inducible form of nitric oxide synthase (iNOS) and reduced the production of collagen.63 This regulation of iNOS is important because excessive levels of iNOS can lead to formation of large amounts of NO that combine with superoxide radicals to form the damaging species peroxynitrite, and can interfere with the protective benefits of other forms of NO synthase.64 These findings suggest that NIR protects against oxidative stress induced by trauma. Finally, an in vitro study of the effects of red light and NIR (700 to 2000 nm) on human RBCs found that NIR significantly protected RBCs against oxidation.65
Since the introduction of low-level laser (light) therapy in 1967, over two hundred randomized, double-blinded, and placebo-controlled phase III clinical trials have been published from over a dozen countries. Whereas there is some degree of consensus as to the best wavelengths of light and acceptable dosages to be used, there is no agreement on whether continuous wave (CW) or pulsed wave (PW) light is more suitable for the various applications of LLLT. This review will raise (but not necessarily answer) several questions. How does pulsed light differ from CW on the cellular and molecular level, and how is the outcome of LLLT affected? If pulsing is more efficacious, then at what pulse parameters is the optimal outcome achieved? In particular, what is the ideal pulse repetition rate or frequency to use?
There are five parameters that could be specified for pulsed light sources. The pulse width or duration or ON time (PD) and the pulse Interval or OFF time (PI) are measured in seconds. Pulse repetition rate or frequency (F) is measured in Hz. The duty cycle (DC) is a unitless fractional number or %. The peak power and average power are measured in Watts.
Pulse duration, pulse repetition rate, and duty cycle are related by the simple equation:
Peak power is a measure of light intensity during the pulse duration, and related to the average power (measured in Watts) by:
In all cases, it is necessary to specify any two out of three of: PD, F, and DC, and either the peak or average power for the pulse parameters to be fully defined.
Figure 1 graphically shows the relationship between peak power and pulse duration.
Five major types of pulsed lasers (or other light sources) are commonly utilized: (1) Q-switched, (2) Gain-switched, (3) Mode-locked, (4) Superpulsed, and (5) Chopped or gated. Each utilizes a different mechanism to generate light in a pulsed as opposed to continuous manner, and vary in terms of pulse repetition rates, energies, and durations. However the first three classes of “truly” pulsed lasers mentioned above are in general not used for LLLT; instead superpulsed or gated lasers are mainly used. The concept of super-pulsing was originally developed for the carbon dioxide laser used in high power tissue ablative procedures. The idea was that by generating relatively short pulses (µsecond) the laser media could be excited to higher levels than those normally allowed in CW mode where heat dissipation constraints limit the maximum amounts of energy that can be used to excite the lasing media. With the original carbon dioxide superpulsed lasers, the short pulses would confine the thermal energy in the tissue (by making the pulse duration less than the thermal diffusion time) reducing collateral thermal damage to normal tissue.
Another type of laser that particularly benefited from super-pulsing is the gallium-arsenide (GaAs) diode laser. This laser has a wavelength in the region of 904-nm and pulse duration usually in the range of 100–200 nanoseconds. Another semiconductor laser amenable to superpulsing is the indium-gallium-arsenide (In-Ga-As) diode laser. It emits light at a similar wavelength (904–905-nm) as the GaAs diode laser, producing very brief pulses (200 nanoseconds) of high frequencies (in the range of kilohertz). These pulses are of very high peak powers (1–50 W) and an average power of 60 mW. Theoretically, the super-pulsed GaAs and In-Ga-As lasers allow for deep penetration without the unwelcome effects of CW (such as thermal damage), as well as allowing for shorter treatment times.
The other major class of pulsed light sources used in LLLT are simply CW lasers (usually diode lasers) that have a pulsed power supply generated by a laser driver containing a pulse generator. This technology is described as “chopped” or “gated.” It is also equally feasible to use pulse generator technology to pulse LEDs or LED arrays .
Pulsed light offers numerous potential benefits. Because there are “quench periods” (pulse OFF times) following the pulse ON times, pulsed lasers can generate less tissue heating. In instances where it is desirable to deliver light to deeper tissues increased powers are needed to provide adequate energy at the target tissue. This increased power can cause tissue heating at the surface layers and in this instance pulsed light could be very useful. Whereas CW causes an increase in temperature of the intervening and target tissues or organ, pulsed light has been shown to cause no measurable change in the temperature of the irradiated area for the same delivered energy density. Anders et al. administered pulsed light to pig craniums, and found no significant change in temperature of the scalp or skull tissue (J.J. Anders, personal communication). Ilic et al.  found that pulsed light (peak power densities of 750 mW/cm2) administered for 120 seconds produced no neurological or tissue damage, whereas an equal power density delivered by CW (for the same number of seconds) caused marked neurological deficits.
Aside from safety advantages, pulsed light might simply be more effective than CW. The “quench period” (pulse OFF times) reduces tissue heating, thereby allowing the use of potentially much higher peak power densities than those that could be safely used in CW. For example, when CW power densities at the skin of ≥2 W/cm2 are used, doubling the CW power density would only marginally increase the treatment depth while potentially significantly increasing the risk of thermal damage; in contrast, peak powers of ≥5 W/cm2 pulsed using appropriate ON and OFF times might produce little, or no tissue heating. The higher peak powers that can be safely used by pulsing light can overcome tissue heating problems and improve the ability of the laser to penetrate deep tissues achieving greater treatment depths.
There may be other biological reasons for the improved efficacy of pulsed light (PW) over CW. The majority of the pulsed light sources used for LLLT have frequencies in the 2.5–10,000 Hz range and pulse durations are commonly in the range of a few millisecond. This observation suggests that if there is a biological explanation of the improved effects of pulsed light it is either due to some fundamental frequency that exists in biological systems in the range of tens to hundreds of Hz, or alternatively due to some biological process that has a time scale of a few milliseconds. Two possibilities for what these biological processes could actually be occur to us. Firstly, it is known that mammalian brains have waves that have specific frequencies . Electroencephalography studies have identified four distinct classes of brain waves [4,5]. Alpha waves (8–13 Hz) occur in adults who have their eyes closed or who are relaxed . Beta waves (14–40 Hz) mainly occur in adults who are awake, alert or focused . Delta waves (1–3 Hz) occur mainly in infants, adults in deep sleep, or adults with brain tumors . Theta waves (4–7 Hz) occur mainly in children ages 2–5 years old and in adults in the twilight state between sleeping and waking or in meditation . The possibility of resonance occurring between the frequency of the light pulses and the frequency of the brain waves may explain some of the results with transcranial LLLT using pulsed light.
Secondly, there are several lines of evidence that ion channels are involved in the subcellular effects of LLLT. Some channels permit the passage of ions based solely on their charge of positive (cationic) or negative (anionic) while others are selective for specific species of ion, such as sodium or potassium. These ions move through the channel pore single file nearly as quickly as the ions move through free fluid. In some ion channels, passage through the pore is governed by a “gate,” which may be opened or closed by chemical or electrical signals, temperature, or mechanical force, depending on the variety of channel. Ion channels are especially prominent components of the nervous system. Voltage-activated ion channels underlie the nerve impulse and while transmitter-activated or ligand-gated channels mediate conduction across the synapses.
There is a lot of literature on the kinetics of various classes of ion channels but in broad summary it can be claimed that the time scale or kinetics for opening and closing of ion channels is of the order of a few milliseconds. For instance Gilboa et al.  used pulses having a width 10 milliseconds and a period of 40 milliseconds (25 Hz). Other reports on diverse types of ion channels have given kinetics with timescales of 160 milliseconds , 3 milliseconds  and one paper giving three values of 0.1, 4 and 100 milliseconds . Potassium and calcium ion channels in the mitochondria and the sarcolemma may be involved in the cellular response to LLLT [14–16].
Thirdly there is the possibility that one mechanism of action of LLLT on a cellular level is the photodissociation of nitric oxide from a protein binding site (heme or copper center) such as those found in cyctochrome c oxidase . If this process occurs it is likely that the NO would rebind to the same site even in the presence of continuous light. Therefore if the light was pulsed multiple photodissociation events could occur, while in CW mode the number of dissociations may be much smaller.
The most important parameter that governs the depth of penetration of laser light into tissue is wavelength. Both the absorption and scattering coefficients of living tissues are higher at lower wavelength so near-infrared light penetrates more deeply that red and so on. It is often claimed that pulsed lasers penetrate more deeply into tissue than CW lasers with the same average power. Why exactly should this be so? Let us suppose that at a certain wavelength (for instance 810-nm) the depth of tissue at which the intensity of a laser is reduced to 10% of its value at the surface of the skin is 1-cm. Therefore if we are using a laser with a power density (irradiance) of 100 mW/cm2 at the skin, the power density remaining at 1 cm below the skin is 10 mW/cm2 and at 2-cm deep is 1 mW/cm2. Now let us suppose that a certain threshold power density (minimum number of photons per unit area per unit time) at the target tissue is necessary to have a biological effect and that this value is 10 mW/cm2. The effective penetration depth at CW may be said to be 1-cm. Now let us suppose that the laser is instead pulsed with a 10-milliseconds pulse duration at a frequency of 1 Hz (DC = 1 Hz×0.010 seconds = 0.010) and the same average power. The peak power and peak power densities are now 100 times higher (peak power = average power/DC = average power×100). With a peak power density of 10 W/cm2 at the skin, the tissue depth—at which this peak power density is attenuated to the threshold level of 10 mW/cm2—is now 3-cm rather than 1-cm in CW mode. But what we have to consider is that the laser is only on for 1% of the time so the total fluence delivered to the 3-cm depth in pulsed mode is 100 times less than that delivered to 1-cm depth in CW mode. However it would be possible to increase the illumination time by a factor of 100 to reach the supposed threshold of fluence as well as the threshold of power at the 3-cm depth. In reality the increase in effective penetration depth obtained with pulsed lasers is more modest than simple calculations might suggest. Many applications of LLLT do not require deep penetration such as tendinopathies and joint pain.
Similarly, deep penetration is often not required to alleviate joint pain. The target tissue in such cases is the synovia; with the exception of back, neck, and hip, most joints have readily accessible synovia. Bjordal et al.  conducted a review of literature and concluded that “superpulsed” lasers (904 nm) were not significantly more effective than CW lasers (810–830 nm); both types of laser achieved similar results, but half the energy was needed to be used for superpulsed lasers. On the other hand, deeper penetrance is needed to reach back, neck, and hip joints. If power densities greater than a few mW/cm2 are to be safely delivered to target tissues >5 cm below the skin, it appears likely that this can only be done by using pulsed lasers. It is postulated that successful LLLT treatments in such joints bring benefit not by reaching the deep target tissue but by inhibiting superficial nociceptors. In other words, they bring relief primarily by attenuating pain perception, as opposed to decreasing inflammation. Does deeper penetration via pulsed lasers offer any significant benefit over CW? It is quite possible that a relatively higher fluence is necessary to attenuate pain, whereas a lower fluence decreases inflammation. If this is indeed the case, for musculo-skeletal applications achieving higher doses at the level of the target tissue may not be ideal. Further studies must be done to confirm this hypothesis, as well as to determine if there is any real benefit to the deeper penetration attained by pulsed lasers. Muscles such as the biceps and rectus femoris are not small organs, and have quite deep target tissue. Yet, various studies have shown significant improvement with CW lasers and CW LED. It remains to be seen whether or not pulsed lasers offer any additional advantage. Similarly, depression  and stroke studies  using LLLT have demonstrated that CW LED’s and CW lasers (respectively) produce a beneficial therapeutic effect. There are reports from Anders’ laboratory that fluences as low as 0.1–0.2 J/cm2 may be optimal for cells in the brain . However, further studies must be done to determine whether pulsed light, with higher peak power densities deeper into the brain tissues, might increase the effectiveness of these therapies.
In this review thirty-three studies involving pulsed LLLT were examined. Of these studies, nine of them directly compared continuous wave (CW) with pulsed wave (PW) light, as recorded in Table 1. Six of these nine studies found PW to be more effective than CW. One study comparing CW and PW found both modes of operation to be equally effective, with no statistically significant difference between the two. Only two of the nine articles reported better results with CW than PW, although in both of these studies PW treated subjects were found to have better outcomes than placebo groups. One of the recurring limitations of the papers in this review was that like for like irradiation parameters were not used. For instance, Gigo-Benato et al.  found CW superior to PW in nerve regeneration, but is this because of the mode of operation (CW or PW) or because the CW laser used 808 nm and the pulsed laser used 905 nm?
Of the six studies that found PW to be more effective than CW, four of them involved the use of LLLT to cure the following pathologies in vivo: wound healing, pain, and ischemic stroke. The two remaining studies reported pulsing to be beneficial in vitro; in the first such study, PW promoted bone stimulation more so than CW. The other in vitro study comparing CW and PW found the latter mode of operation better able to penetrate through melanin filters, indicating that pulsing may be beneficial in reaching deep target tissue in dark-skinned patients.
In the wound healing study, Kymplova et al.  used a large sample size of women to study the effects of phototherapy on wound repair following surgical episiotomies (one of the most common surgical procedures in women). A pulsed laser emitted light (wavelength of 670 nm) at various frequencies (10, 25, and 50 Hz). The pulsed laser promoted wound repair and healing more so than the CW light source.
In the pain study, Sushko et al.  investigated the role of pulsed LLLT to attenuate pain in white male mice. The same wavelength of light was used as in Kymplova et al.’s study (670 nm), with the frequencies of 10, 600, and 8,000 Hz. Both modes of delivery (CW and PW) reduced the behavioral manifestations of somatic pain as compared to controls, but pulsed light (10 and 8,000 Hz in particular) was more effective.
The two studies involving pulsed LLLT and stroke were both done by Lapchak et al. . Ischemic strokes were induced in rabbits, and a pulsed laser with a wavelength of 808 nm was used. In the first study, two frequencies of pulsed light were used (100 and 1,000 Hz), both of which reduced neurological deficits more so than CW. Accordingly, pulsed LLLT may play a major role in the management of stroke patients. Lapchak et al.’s second study attempted to prove the hypothesis that LLLT’s neuroprotective effect following stroke was a result of enhanced mitochondrial energy production (increased ATP synthesis) . As with the previous study, LLLT was administered following stroke induction. CW radiation raised cortical ATP levels but was unable to bring them back to baseline. PW radiation, on the other hand, not only mitigated the effects of stroke on cortical ATP levels, but was able to raise cortical ATP levels to higher than those found in healthy rabbits (those in which stroke was not induced). This study provides valuable insight into one of the potential cellular and molecular mechanisms behind the enhanced neurogenesis (and improved clinical outcomes) observed in subjects receiving transcranial LLLT following stroke.
One of the nine studies reviewed found CW and PW to be equally effective in the promotion of wound healing. This study compared the effects of a CW laser (632.8 nm) and a PW laser (904 nm) on the promotion of wound healing in rabbits. Both lasers improved tensile strength during wound healing, but did not significantly improve wound-healing rates. A combined laser (CW+PW) was also tested. All three of the laser regimens improved tensile strength to a similar extent.
As mentioned earlier, there were nine studies that compared CW and PW, only two of which found CW to be more effective. These two studies involved wound healing and nerve regeneration respectively. Al-Watban and Zhang  study involved rats that were inflicted with aseptic wounds. The rats were divided into three groups: a control group, those irradiated with continuous wave light, and those irradiated with pulsed light at various repetition rates (100, 200, 300, 400, and 500 Hz). Of the pulse repetition rates administered, 100 Hz was the most efficacious and 500 Hz the least. Both CW and PW (635 nm) promoted wound healing, but CW was more efficacious. These results conflict with earlier studies that found pulsed light to be more beneficial in the promotion of wound healing. However, it should be noted that the difference between CW and PW treated subjects was small (a relative wound healing rate of 4.81 as compared to 4.32).
The second study that found CW to be more effective than PW involved nerve regeneration. There were three articles involving nerve regeneration, all of which found pulsed LLLT to be ineffective, as discussed in the section below entitled “Studies Involving Nerve Conduction and Regeneration.” Of these three, only Gigo-Benato et al.  compared CW (808 nm) and PW (905 nm). This study involved rats in which the left median nerve was completely transected and then repaired by end-to-end neurorrhaphy. The CW laser (808 nm) promoted faster nerve and muscle recovery than the pulsed laser (905 nm). However, Gigo-Benato also tested a combination of the CW and pulsed lasers, finding this to be the most effective of all. In other words, seven of the nine studies comparing CW and PW found pulsing to play a beneficial role. Only one of the nine studies found no role of PW, and even in this study the benefit of CW over PW was minimal.
We reviewed three studies, as recorded in Table 2, which investigated the role of a combined laser (using both CW and PW). Of these, only Gigo-Benato’s study compared the combined laser to stand alone CW or PW. This study has been discussed in the above section: the combined laser was found to be effective in stimulating nerve regeneration, more so than CW or PW alone.
The two other studies used a combined laser (CW and PW) to administer laser acupuncture, along with Transcutaneous Electrical Nerve Stimulation (TENS), to patients with symptoms of pain. Naeser et al.  administered this “triple therapy” to patients suffering from carpal tunnel syndrome (CTS). Eleven patients with mild-to-moderate symptoms of CTS were selected, all of who had failed to respond to standard medical or surgical treatment regimens. Subjects were divided into two groups, one of which received sham irradiation and the other that received a combined treatment of LLLT (CW and pulsed) and TENS. As compared to controls, the treated group experienced statistically significant improvement and remained stable for 1–3 years. The results of this study are promising, and indicate a possible role of LLLT and TENS in the conservative management of CTS.
Ceccherelli et al.  administered laser acupuncture to patients suffering from myofascial pain. In this double-blinded placebo controlled trial, patients received either the same “triple therapy” as in the Naeser et al. study (CW, PW, and TENS) or sham irradiation, every other day over the course of 24 days. Results were encouraging, with the treatment group experiencing a significant improvement in symptoms, both immediately after the treatment regimen and at a 3-month follow up visit.
In both preceding studies, the combined regimen of CW, PW, and TENS was compared to untreated controls, and found to be effective. However, neither study compared CW and PW or administered CW, PW, or TENS individually. As such, it is difficult to determine whether standalone CW or PW would have produced similar results, or if the combined regimen (along with TENS) was necessary.
Of the 33 studies reviewed, 21 of them compared PW treated subjects with untreated controls, as reported in Table 3. Of these, fourteen studies found pulsed LLLT to be effective, whereas seven of them found PW treated subjects to have no benefit over untreated controls. Only one study found PW to have a worse outcome than controls. Of the fourteen studies that found pulsed LLLT to be effective, seven involved the promotion of wound healing, four involved the attenuation of pain, two involved the promotion of bone and cartilage growth respectively, and one involved the treatment of a very rare condition (hyperphagic syndrome caused by traumatic brain injury). Of the seven studies that found no benefit to pulsed light, three involved the promotion of nerve conduction, two involved the promotion of nerve regeneration, and the remaining two involved the attenuation of pain.
If pulsed LLLT is effective (or ineffective), then what pulse repetition rates are to be used (or avoided)? Ten of the 33 articles reviewed tested and compared various repetition rates, as reported in Table 4. Four of these studies involved the use of pulsed LLLT to promote wound healing. Longo et al.  used the pulse repetition rates of 1,500 and 3,000 Hz, and found only the latter setting to promote wound healing. Korolev et al.  similarly used two pulse repetition rates, 500 and 3,000 Hz. In this case, both were found to be effective but 500 Hz was more so. Al-Watban and Zhang  compared five different pulse repetition rates (100, 200, 300, 400 and 500 Hz), finding 100 Hz to be the most effective and 500 Hz the least. el Sayed and Dyson  compared four different pulse repetition rates (2.5, 20, 292, and 20,000 Hz), and found only the two middle values (20 and 292 Hz) beneficial. The more effective pulse repetition rates in these four studies were very disparate, including 20, 100, 292, 500, and 3,000 Hz (a range of 20–3,000 Hz).
Two studies compared the role of various pulse repetition rates in the attenuation of pain. Ponnudurai et al.  used laser photobiostimulation to decrease pain levels in rats, and investigated the effect of using various pulsing frequencies (4, 60, and 200 Hz). The rat tail-flick test was utilized, and tail-flick latencies were measured at five intervals between 30 minutes and 7 days following irradiation. The pulsing frequency of 4 Hz increased pain threshold rapidly but very transiently, whereas 60 Hz produced a delayed but longer lasting effect. On the other hand, 200 Hz failed to produce any hypoalgesic effect whatsoever. Sushko et al.  conducted a similar experiment, using mice instead of rats. The center of pain was irradiated (610–910 nm) for 10 minutes with either CW or pulsed light (10, 600, and 8,000 Hz). Both modes of delivery (CW and pulsed) reduced the behavioral manifestations of somatic pain as compared to controls, but pulsed light was more effective. In particular, 10 and 8,000 Hz produced the best effect. The more effective pulse repetition rates from these two studies (involving pain attenuation) included 4, 10, 60, and 8,000 Hz (a range of 4–8,000 Hz), and the less effective pulse repetition rates included 200 and 600 Hz.
Lapchak et al.  not only compared CW and PW, but also pulsed light at two different repetition rates, P1 (1,000 Hz) and P2 (100 Hz). Ischemic strokes were induced in rabbits, and the neuroprotective effects of LLLT were assessed via behavioral analysis 48 hours post-stroke. Both P1 (1,000 Hz) and P2 (100 Hz) produced a similar effect (superior to CW).
Rezvani et al.  studied the use of low level light therapy to prevent X-ray induced late dermal necrosis. An X-ray dose of 23.4 Gy is known to invariably cause dermal necrosis after 10–16 weeks. This dose was delivered to pigs, which were then treated with LLLT for several weeks using various wavelengths (660, 820, 880, and 950 nm) pulsed at either 2.5 or 5,000 Hz. Light pulsed at 2.5 Hz did not reduce the incidence of dermal necrosis. On the other hand, light pulsed at 5,000 Hz significantly reduced (P = 0.001) the incidence to 52% when given 6–16 weeks after irradiation.
Of the 10 articles reviewed that compared various pulse repetition rates, two of them involved in vitro experiments. Brondon et al.  undertook a study to determine if pulsing light would overcome the filtering effects of melanin. Melanin filters were placed in front of human HEP-2 cells, which were then irradiated for 72 hours (670 nm wavelength) with either CW or pulsed light at various repetition rates (6, 18, 36, 100, and 600 Hz). Both cell proliferation and oxidative burst activity, were increased in the group treated with pulsed light, indicating that pulsed light is indeed better able to penetrate melanin rich skin. Specifically, cell proliferation was maximal at 100 Hz at 48 and 72 hours (n = 4, P≤0.05), and oxidative burst was maximal at 600 Hz (n = 4, P≤0.05).
Ueda and Shimizu  studied the effects of pulsed low-level light on bone formation in vitro. Osteoblast-like cells were isolated from fetal rat calvariae; one group was not irradiated at all, another was irradiated with continuous wave light, and the third group with pulsed light at three repetition rates (1, 2, and 8 Hz). As compared to the control group, both CW and PW light resulted in increased cellular proliferation, bone nodule formation, alkaline phosphatase (ALP) gene expression, and ALP activity. Pulsed light at 2 Hz stimulated these factors the most.
Out of all 10 articles that compared various pulse repetition rates, the following pulse repetition rates were found to be beneficial: 2, 10, 20, 100, 292, 500, 600, 1,000, 3,000, 5,000, and 8,000 Hz. In this wide range of frequencies (2–8,000 Hz), no particular frequencies stood out as being particularly more or less useful than others.
Ten studies out of the 33 involved LLLT’s role in the promotion of wound healing, as recorded in Table 5. Only two of these studies compared CW and PW. Kymplova et al.  found pulsed LLLT to promote wound healing over CW, whereas Al-Watban and Zhang  found CW to be slightly more effective than PW. Both studies used light of a similar wavelength (670 vs. 635 nm), although the pulse repetition rates used by Kymplova et al. were lower (10–50 Hz vs. 100–500 Hz in Al-Watban et al.’s study). The energy densities applied were also different (2 J/cm2 vs. 1 J/cm2).
Every study reviewed found pulsed LLLT effective in promoting wound healing (as compared to untreated controls), including the Al-Watban et al. study. Six of these studies used light in the wavelength range of 820–956 nm, and four in the range of 632.8–670 nm. Once again, a wide range of frequencies were used (2.5–20,000 Hz), most of which were found to promote wound healing. (Tested frequencies included 2.5, 5, 8.58, 10, 15.6, 20, 25, 31.2, 50, 78, 80, 287, 292, 500, 700, 3,000, 4,672, 9,000, and 20,000 Hz). Most of these articles also reported energy densities, usually in the range of 1–2 J/cm2.
We reviewed three articles evaluating the role of pulsed LLLT in the promotion of nerve conduction, and another three involving nerve regeneration, as reported in Table 6. Unlike the studies involving wound healing where positive outcomes were reported, all six of these studies reported negative outcomes with pulsed light. Five of these studies found PW to have no statistically significant effect on outcome, whereas one of them found PW to have a deleterious effect. There was no study that directly compared CW and PW in regards to nerve conduction. Walsh et al.  conducted a study with 32 human volunteers to determine if pulsed LLLT would influence nerve conduction in the superficial radial nerve. Action potentials were measured pre- and post-irradiation (at 5, 10, and 15 minutes). No significant difference was appreciated between control and treatment groups, indicating that LLLT with those particular pulsing parameters and dosimetry had no specific neurophysiologic effects on nerve conduction. Bagis et al.  and Comelekoglu et al.  obtained similar negative results using frog nerves. Walsh et al. used a wavelength of 820 nm, whereas Bagis et al. used a 904 nm laser. All three studies tested pulse repetition rates within the range of 1–128 Hz.
Similarly, the nerve regeneration studies reviewed reported negative outcomes. Chen et al.  found PW to have a counterproductive effect, reducing nerve regeneration as compared to untreated controls. Only one study compared CW with PW, and found the former to be superior to the latter. However, the combined laser (CW+PW) was superior to CW alone, indicating that there might in fact be a role of pulsing in nerve regeneration.
Nine of the thirty-three studies involved pulsed LLLT’s role in the attenuation of pain, as reported in Table 7. Of these, only one of them directly compared CW and PW. This study was conducted by Sushko et al.  and found that although both CW and PW decreased pain levels, PW was more effective. This study also determined that pulse repetition rates of 10 and 8,000 Hz were more effective than 600 Hz. Ponnudurai et al.  similarly compared various pulse repetition rates (4, 60, and 200 Hz). A rapid but transient analgesic effect was exhibited with 4 Hz, whereas a delayed but longer lasting effect was achieved with 60 Hz. On the other hand, 200 Hz failed to produce any analgesic effect whatsoever.
Two of the studies used a combined laser (CW+PW) along with TENS; both found the combined regimen to be effective. The five remaining studies compared pulsed LLLT with untreated controls. Three of these studies found pulsed LLLT to be effective, whereas two did not. Of the nine total studies on pain attenuation, seven found pulsed LLLT to be effective in its role of attenuating pain. Only two studies found no statistically significant effect. However, it should be noted that both of these involved pain of a different nature than commonly tested in pulsed LLLT studies. The first of these was by Craig et al.  and involved the use of pulsed LLLT to relieve the symptoms of delayed-onset muscle soreness (DOMS). DOMS refers to the feeling of pain and muscle stiffness that can result 1–3 days after intense sporting activity such as weightlifting. This pain is duller in quality than that tested in the other studies. The second study that showed no benefit to pulsed LLLT, published by de Bie et al. , involved the treatment of lateral ankle sprains.
Table 8 records the two studies that involved pulsed LLLT and stroke. In the first study, PW but not CW decreased neurological deficits when delivered six hours post-stroke. Two pulse repetition rates were tested (100 and 1,000 Hz) and found to be equally effective. On the other hand, both CW and PW produced no benefit if delivered 12 hours post-stroke, indicating that timely administration of LLLT is essential.
The second study investigated the possible mechanisms behind the neuroprotective effect of LLLT. It was postulated that LLLT enhances mitochondrial energy production (and ATP synthesis), which allows for enhanced neurogenesis. This hypothesis was tested using the rabbit small clot embolic stroke model (RSCEM). Four groups of rabbits were used: (1) a naïve control group which was neither embolized or irradiated, (2) a placebo group which was embolized and sham irradiated, (3) an embolized group which was irradiated with CW (808 nm), and (4) an embolized group which was irradiated with pulsed light (808 nm) at two different frequencies. Forty-five percent less cortical ATP was measured in the second group (placebo) as compared to the first (naïve), confirming the hypothesis that ischemic strokes decrease cortical mitochondrial energy. All laser irradiated groups were able to mitigate this effect. CW radiation managed to raise the cortical ATP levels by 41%, whereas PW administration raised these levels by over 150%. Surprisingly, this was even higher than the cortical ATP content measured in naïve rabbits that had never suffered stroke.
Many of the modalities of treatment employed in biomedicine and physical therapy are used in pulsed format . Electricity, electromagnetic fields and ultrasound are applied with particular pulse structures. It may be possible to gain some insight into the effect of pulsing structures in LLLT by a brief review of the other pulsed modalities. Transcutaneous electrical neural stimulation (TENS) is the application of pulses of electric current to the skin . This application stimulates the brain and has been used for the treatment of various psychological and neurological conditions, including Parkinson’s, epilepsy, chronic pain, depression, and neuromuscular rehabilitation. Frequencies usually fall between 5 and 25 Hz, but may range from 2 to 80 Hz . Deep brain stimulation (DBS) is a surgical treatment involving the implantation of a brain pacemaker, a medical device that sends electrical impulses to specific parts of the brain. DBS has the potential to provide substantial benefit to patients suffering from a variety of neurological conditions, including epilepsy, Parkinson’s disease, dystonia, Tourette’s syndrome, and depression . The Food and Drug Administration (FDA) approved DBS at 130 Hz as a treatment for essential tremor in 1997, for Parkinson’s disease in 2002, and dystonia in 2003. Pulsed electromagnetic field (PEMF) therapy has been used for a wide range of conditions, including bone healing and regeneration , osteoporosis , arthritis  wound healing and pain , carpal tunnel syndrome , spinal cord injury , nerve regeneration , soft tissue injuries , and cancer . Frequencies used for these conditions range from 1 Hz (“low”) to 200 Hz (“high”). Transcranial magnetic stimulation (TMS) is a noninvasive method used to excite neurons in the brain. Weak electric currents are induced by butterfly coils positioned above the head. TMS has been approved for the treatment of resistant depression in several countries and is under investigation for migraine , aphasia , and tinnitus . Low-intensity pulsed ultrasound (LIPUS) utilizes a non-thermal mechanism of action, which can be used to promote bone healing by inducing the expression of growth factors and prostaglandins, which stimulate osteoblasts, chondrocytes and fibroblasts .
There has been remarkably little information available in the peer-reviewed literature on the rationale for using pulsed lasers or pulsed light in LLLT rather than CW. Moreover there is no consensus on the effects of different frequencies and pulse parameters on the physiology and therapeutic response of the various disease states that are often treated with laser therapy. This has allowed manufacturers to claim advantages of pulsing without hard evidence to back up their claims.
CW light is the gold standard and has been used for all LLLT applications. However, this review of the literature indicates that overall pulsed light may be superior to CW light with everything else being equal. This seemed to be particularly true for wound healing and post-stroke management. On the other hand, PW as a solo treatment may be less beneficial than CW in patients requiring nerve regeneration. This could possibly be explained by the mechanism of action LLLT that can either cause cell stimulation or cell inhibition or both stimulation and inhibition at the same time on different cell types. It is possible that stimulation in neurons is desired to promote neurogenesis following stroke (increased mitochondrial synthesis of ATP results in more energy for neurons to regenerate themselves), whereas inhibition of inflammatory cells, inhibition of immune response or inhibition of the glial scar may also occur at the same time. The logic in favor of PW is that cells may need periods of rest, without which they can no longer be stimulated further.
Considering that the biology of LLLT is known to be complex, it is likely that there may several optimal sets of pulse parameters and that these may relate to the specific wavelengths and chromophores and may well also be affected by other optical properties of tissues.
It was impossible to draw any meaningful correlations between pulse frequency and pathological condition, due to the wide-ranging and disparate data. As for other pulse parameters, these were in general poorly and inconsistentl
Photobiomodulation (PBM) describes the use of red or near-infrared light to stimulate, heal, regenerate, and protect tissue that has either been injured, is degenerating, or else is at risk of dying. One of the organ systems of the human body that is most necessary to life, and whose optimum functioning is most worried about by humankind in general, is the brain. The brain suffers from many different disorders that can be classified into three broad groupings: traumatic events (stroke, traumatic brain injury, and global ischemia), degenerative diseases (dementia, Alzheimer's and Parkinson's), and psychiatric disorders (depression, anxiety, post traumatic stress disorder). There is some evidence that all these seemingly diverse conditions can be beneficially affected by applying light to the head. There is even the possibility that PBM could be used for cognitive enhancement in normal healthy people. In this transcranial PBM (tPBM) application, near-infrared (NIR) light is often applied to the forehead because of the better penetration (no hair, longer wavelength). Some workers have used lasers, but recently the introduction of inexpensive light emitting diode (LED) arrays has allowed the development of light emitting helmets or “brain caps”. This review will cover the mechanisms of action of photobiomodulation to the brain, and summarize some of the key pre-clinical studies and clinical trials that have been undertaken for diverse brain disorders.
Photobiomodulation (PBM) as it is known today (the beneficial health benefits of light therapy had been known for some time before), was accidently discovered in 1967, when Endre Mester from Hungary attempted to repeat an experiment recently published by McGuff in Boston, USA . McGuff had used a beam from the recently discovered ruby laser , to destroy a cancerous tumor that had been experimentally implanted into a laboratory rat. However (unbeknownst to Mester) the ruby laser that had been built for him, was only a tiny fraction of the power of the laser that had previously been used by McGuff. However, instead of curing the experimental tumors with his low-powered laser, Mester succeeded in stimulating hair regrowth and wound healing in the rats, in the sites where the tumors had been implanted , . This discovery led to a series of papers describing what Mester called “laser biostimulation”, and soon became known as “low level laser therapy” (LLLT) , , .
LLLT was initially primarily studied for stimulation of wound healing, and reduction of pain and inflammation in various orthopedic conditions such as tendonitis, neck pain, and carpal tunnel syndrome . The advent of light emitting diodes (LED) led to LLLT being renamed as “low level light therapy”, as it became more accepted that the use of coherent lasers was not absolutely necessary, and a second renaming occurred recently  when the term PBM was adopted due to uncertainties in the exact meaning of “low level”.
The most well studied mechanism of action of PBM centers around cytochrome c oxidase (CCO), which is unit four of the mitochondrial respiratory chain, responsible for the final reduction of oxygen to water using the electrons generated from glucose metabolism . The theory is that CCO enzyme activity may be inhibited by nitric oxide (NO) (especially in hypoxic or damaged cells). This inhibitory NO can be dissociated by photons of light that are absorbed by CCO (which contains two heme and two copper centers with different absorption spectra) . These absorption peaks are mainly in the red (600–700 nm) and near-infrared (760–940 nm) spectral regions. When NO is dissociated, the mitochondrial membrane potential is increased, more oxygen is consumed, more glucose is metabolized and more ATP is produced by the mitochondria.
It has been shown that there is a brief increase in reactive oxygen species (ROS) produced in the mitochondria when they absorb the photons delivered during PBM. The idea is that this burst of ROS may trigger some mitochondrial signaling pathways leading to cytoprotective, anti-oxidant and anti-apoptotic effects in the cells . The NO that is released by photodissociation acts as a vasodilator as well as a dilator of lymphatic flow. Moreover NO is also a potent signaling molecule and can activate a number of beneficial cellular pathways . Fig. 2 illustrates these mechanisms.
It is quite clear that there must be some other type of photoacceptor, in addition to CCO, as is clearly demonstrated by the fact that wavelengths substantially longer than the red/NIR wavelengths discussed above, can also produce beneficial effects is some biological scenarios. Wavelengths such as 980 nm , , 1064 nm laser , and 1072 nm LED , and even broad band IR light  have all been reported to carry out PBM type effects. Although the photoacceptor for these wavelengths has by no means been conclusively identified, the leading hypothesis is that it is primarily water (perhaps nanostructured water) located in heat or light sensitive ion channels. Clear changes in intracellular calcium can be observed, that could be explained by light-mediated opening of calcium ion channels, such as members of the transient receptor potential (TRP) super-family . TRP describes a large family of ion channels typified by TRPV1, recently identified as the biological receptor for capsaicin (the active ingredient in hot chili peppers) . The biological roles of TRP channels are multifarious, but many TRP channels are involved in heat sensing and thermoregulation .
Most authors suggest that the beneficial effects of tPBM on the brain can be explained by increases in cerebral blood flow, greater oxygen availability and oxygen consumption, improved ATP production and mitochondrial activity , , . However there are many reports that a brief exposure to light (especially in the case of experimental animals that have suffered some kind of acute injury or traumatic insult) can have effects lasting days, weeks or even months . This long-lasting effect of light can only be explained by activation of signaling pathways and transcription factors that cause changes in protein expression that last for some considerable time. The effects of PBM on stimulating mitochondrial activity and blood flow is of itself, unlikely to explain long-lasting effects. A recent review listed no less than fourteen different transcription factors and signaling mediators, that have been reported to be activated after light exposure .
Fig. 1 illustrates two of the most important molecular photoreceptors or chromophores (cytochrome c oxidase and heat-gated ion channels) inside neuronal cells that absorb photons that penetrate into the brain. The signaling pathways and activation of transcription factors lead to the eventual effects of PBM in the brain.
Fig. 2 illustrates some more tissue specific mechanisms that lead on from the initial photon absorption effects explained in Fig. 1. A wide variety of processes can occur that can benefit a correspondingly wide range of brain disorders. These processes can be divided into short-term stimulation (ATP, blood flow, lymphatic flow, cerebral oxygenation, less edema). Another group of processes center around neuroprotection (upregulation of anti-apoptotic proteins, less excitotoxity, more antioxidants, less inflammation). Finally a group of processes that can be grouped under “help the brain to repair itself” (neurotrophins, neurogenesis and synaptogenesis).
The biphasic dose response (otherwise known as hormesis, and reviewed extensively by Calabrese et al. ) is a fundamental biological law describing how different biological systems can be activated or stimulated by low doses of any physical insult or chemical substance, no matter how toxic or damaging this insult may be in large doses. The most well studied example of hormesis is that of ionizing radiation, where protective mechanisms are induced by very low exposures, that can not only protect against subsequent large doses of ionizing radiation, but can even have beneficial effects against diseases such as cancer using whole body irradiation .
There are many reports of PBM following a biphasic dose response (sometimes called obeying the Arndt-Schulz curve , . A low dose of light is beneficial, but raising the dose produces progressively less benefit until eventually a damaging effect can be produced at very high light . It is often said in this context that “more does not mean more”.
Another question that arises in the field of PBM is whether the coherent monochromatic lasers that were used in the original discovery of the effect, and whose use continued for many years, are superior to the rather recent introduction of LEDs, that are non-coherent and have a wider band-spread (generally 30 nm full-width half-maximum). Although there are one or two authors who continue to believe that coherent lasers are superior , most commentators feel that other parameters such as wavelength, power density, energy density and total energy are the most important determinants of efficacy .
Due to the growing interest in PBM of the brain, several tissue optics laboratories have investigated the penetration of light of different wavelengths through the scalp and the skull, and to what depths into the brain this light can penetrate. This is an intriguing question to consider, because at present it is unclear exactly what threshold of power density in mW/cm2 is required in the b5rain to have a biological effect. There clearly must be a minimum value below which the light can be delivered for an infinite time without doing anything, but whether this is in the region of μW/cm2 or mW/cm2 is unknown at present.
Functional near-infrared spectroscopy (fNIRS) using 700–900 nm light has been established as a brain imaging technique that can be compared to functional magnetic resonance imaging (fMRI) . Haeussinger et al. estimated that the mean penetration depth (5% remaining intensity) of NIR light through the scalp and skull was 23:6 + 0:7 mm . Other studies have found comparable results with variations depending on the precise location on the head and wavelength , .
Jagdeo et al.  used human cadaver heads (skull with intact soft tissue) to measure penetration of 830 nm light, and found that penetration depended on the anatomical region of the skull (0.9% at the temporal region, 2.1% at the frontal region, and 11.7% at the occipital region). Red light (633 nm) hardly penetrated at all. Tedord et al.  also used human cadaver heads to compare penetration of 660 nm, 808 nm, and 940 nm light. They found that 808 nm light was best and could reach a depth in the brain of 40–50 mm. Lapchak et al. compared the transmission of 810 nm light through the skulls of four different species, and found mouse transmitted 40%, while for rat it was 21%, rabbit it was 11.3 and for human skulls it was only 4.2% . Pitzschke and colleagues compared penetration of 670 nm and 810 nm light into the brain when delivered by a transcranial or a transphenoidal approach, and found that the best combination was 810 nm delivered transphenoidally . In a subsequent study these authors compared the effects of storage and processing (frozen or formalin-fixed) on the tissue optical properties of rabbit heads . Yaroslavsky et al. examined light penetration of different wavelengths through different parts of the brain tissue (white brain matter, gray brain matter, cerebellum, and brainstem tissues, pons, thalamus). Best penetration was found with wavelengths between 1000 and 1100 nm .
Henderson and Morries found that between 0.45% and 2.90% of 810 nm or 980 nm light penetrated through 3 cm of scalp, skull and brain tissue in ex vivo lamb heads .
It is in fact very likely that the beneficial effects of PBM on the brain cannot be entirely explained by penetration of photons through the scalp and skull into the brain itself. There have been some studies that have explicitly addressed this exact issue. In a study of PBM for Parkinson's disease in a mouse model . Mitrofanis and colleagues compared delivering light to the mouse head, and also covered up the head with aluminum foil so that they delivered light to the remainder of the mouse body. They found that there was a highly beneficial effect on neurocognitive behavior with irradiation to the head, but nevertheless there was also a statistically significant (although less pronounced benefit, referred to by these authors as an ‘abscopal effect”) when the head was shielded from light . Moreover Oron and co-workers  have shown that delivering NIR light to the mouse tibia (using either surface illumination or a fiber optic) resulted in improvement in a transgenic mouse model of Alzheimer's disease (AD). Light was delivered weekly for 2 months, starting at 4 months of age (progressive stage of AD). They showed improved cognitive capacity and spatial learning, as compared to sham-treated AD mice. They proposed that the mechanism of this effect was to stimulate c-kit-positive mesenchymal stem cells (MSCs) in autologous bone marrow (BM) to enhance the capacity of MSCs to infiltrate the brain, and clear β-amyloid plaques . It should be noted that the calvarial bone marrow of the skull contains substantial numbers of stem cells .
Laser acupuncture is often used as an alternative or as an addition to traditional Chinese acupuncture using needles . Many of the applications of laser acupuncture have been for conditions that affect the brain  such as Alzheimer's disease  and autism  that have all been investigated in animal models. Moreover laser acupuncture has been tested clinically .
A wide array of different light sources (lasers and LEDs) have been employed for tPBM. One of the most controversial questions which remains to be conclusively settled, is whether a coherent monochromatic laser is superior to non-coherent LEDs typically having a 30 nm band-pass (full width half maximum). Although wavelengths in the NIR region (800–1100 nm) have been the most often used, red wavelengths have sometimes been used either alone, or in combination with NIR. Power levels have also varied markedly from Class IV lasers with total power outputs in the region of 10 W , to lasers with more modest power levels (circa 1 W). LEDs can also have widely varying total power levels depending on the size of the array and the number and power of the individual diodes. Power densities can also vary quite substantially from the Photothera laser  and other class IV lasers , which required active cooling (~ 700 mW/cm2) to LEDs in the region of 10–30 mW/cm2.
One question that is always asked in biomedical research, is how closely do the laboratory models of disease (which are usually mice or rats) mimic the human disease for which new treatments are being sought? This is no less critical a question when the areas being studied include brain disorders and neurology. There now exist a plethora of transgenic mouse models of neurological disease , . However in the present case, where the proposed treatment is almost completely free of any safety concerns, or any reported adverse side effects, it can be validly questioned as to why the use of laboratory animal models should be encouraged. Animal models undoubtedly have disadvantages such as failure to replicate all the biological pathways found in human disease, difficulty in accurately measuring varied forms of cognitive performance, small size of mice and rats compared to humans, short lifespan affecting the development of age related diseases, and lack of lifestyle factors that adversely affect human diseases. Nevertheless, small animal models are less expensive, and require much less time and effort to obtain results than human clinical trials, so it is likely they will continue to be used to test tPBM for the foreseeable future.
Perhaps the most well-investigated application of PBM to the brain, lies in its possible use as a treatment for acute stroke . Animal models such as rats and rabbits, were first used as laboratory models, and these animals had experimental strokes induced by a variety of methods and were then treated with light (usually 810 nm laser) within 24 h of stroke onset . In these studies intervention by tLLLT within 24 h had meaningful beneficial effects. For the rat models, stroke was induced by middle cerebral artery occlusion (MCAO) via an insertion of a filament into the carotid artery or via craniotomy , . Stroke induction in the “rabbit small clot embolic model” (RSCEM) was by injection of a preparation of small blood clots (made from blood taken from a second donor rabbit) into a catheter placed in the right internal carotid artery . These studies and the treatments and results are listed in Table 1.
CW, continuous wave; LLLT, low level light therapy; MCAO, middle cerebral artery occlusion; NOS, nitric oxide synthase; RSCEM, rabbit small clot embolic model; TGFβ1, transforming growth factor β1.
Treatment of acute stroke was addressed in a series of three clinical trials called “Neurothera Effectiveness and Safety Trials” (NEST-1 , NEST-2 , and NEST-3 ) using an 810 nm laser applied to the shaved head within 24 h of patients suffering an ischemic stroke. The first study, NEST-1, enrolled 120 patients between the ages of 40 to 85 years of age with a diagnosis of ischemic stroke involving a neurological deficit that could be measured. The purpose of this first clinical trial was to demonstrate the safety and effectiveness of laser therapy for stroke within 24 h . tPBM significantly improved outcome in human stroke patients, when applied at ~ 18 h post-stroke, over the entire surface of the head (20 points in the 10/20 EEG system) regardless of stroke . Only one laser treatment was administered, and 5 days later, there was significantly greater improvement in the Real- but not in the Sham-treated group (p < 0.05, NIH Stroke Severity Scale). This significantly greater improvement was still present at 90 days post-stroke, where 70% of the patients treated with Real-LLLT had a successful outcome, while only 51% of Sham-controls did. The second clinical trial, NEST-2, enrolled 660 patients, aged 40 to 90, who were randomly assigned to one of two groups (331 to LLLT, 327 to sham) . Beneficial results (p < 0.04) were found for the moderate and moderate-severe (but not for the severe) stroke patients, who received the Real laser protocol . These results suggested that the overall severity of the individual stroke should be taken into consideration in future studies, and very severe patients are unlikely to recover with any kind of treatment. The last clinical trial, NEST-3, was planned for 1000 patients enrolled. Patients in this study were not to receive tissue plasminogen activator, but the study was prematurely terminated by the DSMB for futility (an expected lack of statistical significance) . NEST-1 was considered successful, even though as a phase 1 trial, it was not designed to show efficacy. NEST-2 was partially successful when the patients were stratified, to exclude very severe strokes or strokes deep within the brain . There has been considerable discussion in the scientific literature on precisely why the NEST-3 trial failed . Many commentators have wondered how could tPBM work so well in the first trial, in a sub-group in the second trial, and fail in the third trial. Lapchak's opinion is that the much thicker skull of humans compared to that of the other animals discussed above (mouse, rat and rabbit), meant that therapeutically effective amounts of light were unlikely to reach the brain . Moreover the time between the occurrence of a stroke and initiation of the PBMT may be an important factor. There are reports in the literature that neuroprotection must be administered as soon as possible after a stroke , . Furthermore, stroke trials in particular should adhere to the RIGOR (rigorous research) guidelines and STAIR (stroke therapy academic industry roundtable) criteria . Other contributory causes to the failure of NEST-3 may have been included the decision to use only one single tPBM treatment, instead of a series of treatments. Moreover, the optimum brain areas to be treated in acute stroke remain to be determined. It is possible that certain areas of the brain that have sustained ischemic damage should be preferentially illuminated and not others.
Somewhat surprisingly, there have not as yet been many trials of PBM for rehabilitation of stroke patients with only the occasional report to date. Naeser reported in an abstract the use of tPBM to treat chronic aphasia in post-stroke patients . Boonswang et al.  reported a single patient case in which PBM was used in conjunction with physical therapy to rehabilitate chronic stroke damage. However the findings that PBM can stimulate synaptogenesis in mice with TBI, does suggest that tPBM may have particular benefits in rehabilitation of stroke patients. Norman Doidge, in Toronto, Canada has described the use of PBM as a component of a neuroplasticity approach to rehabilitate chronic stroke patients .
There have been a number of studies looking at the effects of PBM in animal models of TBI. Oron's group was the first  to demonstrate that a single exposure of the mouse head to a NIR laser (808 nm) a few hours after creation of a TBI lesion could improve neurological performance and reduce the size of the brain lesion. A weight-drop device was used to induce a closed-head injury in the mice. An 808 nm diode laser with two energy densities (1.2–2.4 J/cm2 over 2 min of irradiation with 10 and 20 mW/cm2) was delivered to the head 4 h after TBI was induced. Neurobehavioral function was assessed by the neurological severity score (NSS). There were no significant difference in NSS between the power densities (10 vs 20 mW/cm2) or significant differentiation between the control and laser treated group at early time points (24 and 48 h) post TBI. However, there was a significant improvement (27% lower NSS score) in the PBM group at times of 5 days to 4 weeks. The laser treated group also showed a smaller loss of cortical tissue than the sham group .
Hamblin's laboratory then went on (in a series of papers ) to show that 810 nm laser (and 660 nm laser) could benefit experimental TBI both in a closed head weight drop model , and also in controlled cortical impact model in mice . Wu et al.  explored the effect that varying the laser wavelengths of LLLT had on closed-head TBI in mice. Mice were randomly assigned to LLLT treated group or to sham group as a control. Closed-head injury (CHI) was induced via a weight drop apparatus. To analyze the severity of the TBI, the neurological severity score (NSS) was measured and recorded. The injured mice were then treated with varying wavelengths of laser (665, 730, 810 or 980 nm) at an energy level of 36 J/cm2 at 4 h directed onto the scalp. The 665 nm and 810 nm groups showed significant improvement in NSS when compared to the control group at day 5 to day 28. Results are shown in Fig. 3. Conversely, the 730 and 980 nm groups did not show a significant improvement in NSS and these wavelengths did not produce similar beneficial effects as in the 665 nm and 810 nm LLLT groups . The tissue chromophore cytochrome c oxidase (CCO) is proposed to be responsible for the underlying mechanism that produces the many PBM effects that are the byproduct of LLLT. COO has absorption bands around 665 nm and 810 nm while it has low absorption bands at the wavelength of 730 nm . It should be noted that this particular study found that the 980 nm did not produce the same positive effects as the 665 nm and 810 nm wavelengths did; nevertheless previous studies did find that the 980 nm wavelength was an active one for LLLT. Wu et al. proposed that these dissimilar results may be due to the variance in the energy level, irradiance, etc. between the other studies and this particular study .
Ando et al.  used the 810 nm wavelength laser parameters from the previous study and varied the pulse modes of the laser in a mouse model of TBI. These modes consisted of either pulsed wave at 10 Hz or at 100 Hz (50% duty cycle) or continuous wave laser. For the mice, TBI was induced with a controlled cortical impact device via open craniotomy. A single treatment with an 810 nm Ga-Al-As diode laser with a power density of 50 mW/m2 and an energy density of 36 J/cm2 was given via tLLLT to the closed head in mice for a duration of 12 min at 4 h post CCI. At 48 h to 28 days post TBI, all laser treated groups had significant decreases in the measured neurological severity score (NSS) when compared to the control (Fig. 4A). Although all laser treated groups had similar NSS improvement rates up to day 7, the PW 10 Hz group began to show greater improvement beyond this point as seen in Fig. 4. At day 28, the forced swim test for depression and anxiety was used and showed a significant decrease in the immobility time for the PW 10 Hz group. In the tail suspension test which measures depression and anxiety, there was also a significant decrease in the immobility time at day 28, and this time also at day 1, in the PW 10 Hz group.
Studies using immunofluorescence of mouse brains showed that tPBM increased neuroprogenitor cells in the dentate gyrus (DG) and subventricular zone at 7 days after the treatment . The neurotrophin called brain derived neurotrophic factor (BDNF) was also increased in the DG and SVZ at 7 days , while the marker (synapsin-1) for synaptogenesis and neuroplasticity was increased in the cortex at 28 days but not in the DG, SVZ or at 7 days  (Fig. 4B). Learning and memory as measured by the Morris water maze was also improved by tPBM . Whalen's laboratory  and Whelan's laboratory  also successfully demonstrated therapeutic benefits of tPBM for TBI in mice and rats respectively.
Zhang et al.  showed that secondary brain injury occurred to a worse degree in mice that had been genetically engineered to lack “Immediate Early Response” gene X-1 (IEX-1) when exposed to a gentle head impact (this injury is thought to closely resemble mild TBI in humans). Exposing IEX-1 knockout mice to LLLT 4 h post injury, suppressed proinflammatory cytokine expression of interleukin (IL)-Iβ and IL-6, but upregulated TNF-α. The lack of IEX-1 decreased ATP production, but exposing the injured brain to LLLT elevated ATP production back to near normal levels.
Dong et al.  even further improved the beneficial effects of PBM on TBI in mice, by combining the treatment with metabolic substrates such as pyruvate and/or lactate. The goal was to even further improve mitochondrial function. This combinatorial treatment was able to reverse memory and learning deficits in TBI mice back to normal levels, as well as leaving the hippocampal region completely protected from tissue loss; a stark contrast to that found in control TBI mice that exhibited severe tissue loss from secondary brain injury.
Margaret Naeser and collaborators have tested PBM in human subjects who had suffered TBI in the past . Many sufferers from severe or even moderate TBI, have very long lasting and even life-changing sequelae (headaches, cognitive impairment, and difficulty sleeping) that prevent them working or living any kind or normal life. These individuals may have been high achievers before the accident that caused damage to their brain . Initially Naeser published a report  describing two cases she treated with PBM applied to the forehead twice a week. A 500 mW continuous wave LED source (mixture of 660 nm red and 830 nm NIR LEDs) with a power density of 22.2 mW/cm2 (area of 22.48 cm2), was applied to the forehead for a typical duration of 10 min (13.3 J/cm2). In the first case study the patient reported that she could concentrate on tasks for a longer period of time (the time able to work at a computer increased from 30 min to 3 h). She had a better ability to remember what she read, decreased sensitivity when receiving haircuts in the spots where LLLT was applied, and improved mathematical skills after undergoing LLLT. The second patient had statistically significant improvements compared to prior neuropsychological tests after 9 months of treatment. The patient had a 2 standard deviation (SD) increase on tests of inhibition and inhibition accuracy (9th percentile to 63rd percentile on the Stroop test for executive function and a 1 SD increase on the Wechsler Memory scale test for the logical memory test (83rd percentile to 99th percentile) .
Naeser et al. then went on to report a case series of a further eleven patients . This was an open protocol study that examined whether scalp application of red and near infrared (NIR) light could improve cognition in patients with chronic, mild traumatic brain injury (mTBI). This study had 11 participants ranging in age from 26 to 62 (6 males, 5 females) who suffered from persistent cognitive dysfunction after mTBI. The participants' injuries were caused by motor vehicle accidents, sports related events and for one participant, an improvised explosive device (IED) blast. tLLLT consisted of 18 sessions (Monday, Wednesday, and Friday for 6 weeks) and commenced anywhere from 10 months to 8 years post-TBI. A total of 11 LED clusters (5.25 cm in diameter, 500 mW, 22.2 mW/cm2, 13 J/cm2) were applied for about 10 min per session (5 or 6 LED placements per set, Set A and then Set B, in each session). Neuropsychological testing was performed pre-LED application and 1 week, 1 month and 2 months after the final treatment. Naeser and colleagues found that there was a significant positive linear trend observed for the Stroop Test for executive function, in trial 2 inhibition (p = 0.004); Stroop, trial 4 inhibition switching (p = 0.003); California Verbal Learning Test (CVLT)-II, total trials 1–5 (p = 0.003); CVLT-II, long delay free recall (p = 0.006). Improved sleep and fewer post-traumatic stress disorder (PTSD) symptoms, if present beforehand, were observed after treatment. Participants and family members also reported better social function and a better ability to perform interpersonal and occupational activities. Although these results were significant, further placebo-controlled studies will be needed to ensure the reliability of this these data .
Henderson and Morries  used a high-power NIR laser (10–15 W at 810 and 980 nm) applied to the head to treat a patient with moderate TBI. The patient received 20 NIR applications over a 2-month period. They carried out anatomical magnetic resonance imaging (MRI) and perfusion single-photon emission computed tomography (SPECT). The patient showed decreased depression, anxiety, headache, and insomnia, whereas cognition and quality of life improved, accompanied by changes in the SPECT imaging.
There was a convincing study  carried out in an AβPP transgenic mouse of AD. tPBM (810 nm laser) was administered at different doses 3 times/week for 6 months starting at 3 months of age. The numbers of Aβ plaques were significantly reduced in the brain with administration of tPBM in a dose-dependent fashion. tPBM mitigated the behavioral effects seen with advanced amyloid deposition and reduced the expression of inflammatory markers in the transgenic mice. In addition, TLT showed an increase in ATP levels, mitochondrial function, and c-fos expression suggesting that there was an overall improvement in neurological function.
There has been a group of investigators in Northern England who have used a helmet built with 1072 nm LEDs to treat AD, but somewhat surprisingly no peer-reviewed publications have described this approach . However a small pilot study (19 patients) that took the form of a randomized placebo-controlled trial investigated the effect of the Vielight Neuro system (see Fig. 5A) (a combination of tPBM and intranasal PBM) on patients with dementia and mild cognitive impairment . This was a controlled single blind pilot study in humans to investigate the effects of PBM on memory and cognition. 19 participants with impaired memory/cognition were randomized into active and sham treatments over 12 weeks with a 4-week no-treatment follow-up period. They were assessed with MMSE and ADAS-cog scales. The protocol involved in-clinic use of a combined transcranial-intranasal PBM device; and at-home use of an intranasal-only PBM device and participants/ caregivers noted daily experiences in a journal. Active participants with moderate to severe impairment (MMSE scores 5–24) showed significant improvements (5-points MMSE score) after 12 weeks. There was also a significant improvement in ADAS-cog scores (see Fig. 5B). They also reported better sleep, fewer angry outbursts and decreased anxiety and wandering. Declines were noted during the 4-week no-treatment follow-up period. Participants with mild impairment to normal (MMSE scores of 25 to 30) in both the active and sham sub-groups showed improvements. No related adverse events were reported.
An interesting paper from Russia  described the use of intravascular PBM to treat 89 patients with AD who received PBM (46 patients) or standard treatment with memantine and rivastigmine (43 patients). The PBM consisted of threading a fiber-optic through a cathéter in the fémoral artery and advancing it to the distal site of the anterior and middle cerebral arteries and delivering 20 mW of red laser for 20–40 min. The PBM group had improvement in cerebral microcirculation leading to permanent (from 1 to 7 years) reduction in dementia and cognitive recovery.
The majority of studies on PBM for Parkinson's disease have been in animal models and have come from the laboratory of John Mitrofanis in Australia . Two basic models of Parkinson's disease were used. The first employed administration of the small molecule (MPTP or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to mice . MPTP was discovered as an impurity in an illegal recreational drug to cause Parkinson's like symptoms (loss of substantia nigra cells) in young people who had taken this drug . Mice were treated with tPBM (670-nm LED, 40 mW/cm2, 3.6 J/cm2) 15 min after each MPTP injection repeated 4 times over 30 h. There were significantly more (35%–45%) dopaminergic cells in the brains of the tPBM treated mice . A subsequent study showed similar results in a chronic mouse model of MPTP-induced Parkinson's disease . They repeated their studies in another mouse model of Parkinson's disease, the tau transgenic mouse strain (K3) that has a progressive degeneration of dopaminergic cells in the substantia nigra pars compacta (SNc) . They went on to test a surgically implanted intracranial fiber designed to deliver either 670 nm LED (0.16 mW) or 670 nm laser (67 mW) into the lateral ventricle of the brain in MPTP-treated mice . Both low power LED and high power laser were effective in preserving SNc cells, but the laser was considered to be unsuitable for long-term use (6 days) due to excessive heat production. As mentioned above, these authors also reported a protective effect of abscopal light exposure (head shielded) in this mouse model . Recently this group has tested their implanted fiber approach in a model of Parkinson's disease in adult Macaque monkeys treated with MPTP . Clinical evaluation of Parkinson's symptoms (posture, general activity, bradykinesia, and facial expression) in the monkeys were improved at low doses of light (24 J or 35 J) compared to high doses (125 J) .
The only clinical report of PBM for Parkinson's disease in humans was an abstract presented in 2010 
This year marks the 50th anniversary of the discovery of the laser. The development of lasers for medical use, which became known as low-level laser therapy (LLLT) or photobiomodulation, followed in 1967. In recent years, LLLT has become an increasingly mainstream modality, especially in the areas of physical medicine and rehabilitation. At first used mainly for wound healing and pain relief, the medical applications of LLLT have broadened to include diseases such as stroke, myocardial infarction, and degenerative or traumatic brain disorders. This review will cover the mechanisms of LLLT that operate both on a cellular and a tissue level. Mitochondria are thought to be the principal photoreceptors, and increased adenosine triphosphate, reactive oxygen species, intracellular calcium, and release of nitric oxide are the initial events. Activation of transcription factors then leads to expression of many protective, anti-apoptotic, anti-oxidant, and pro-proliferation gene products. Animal studies and human clinical trials of LLLT for indications with relevance to neurology, such as stroke, traumatic brain injury, degenerative brain disease, spinal cord injury, and peripheral nerve regeneration, will be covered.
It was not long after the discovery of the first lasers (the ruby laser in 1960 and the helium-neon [HeNe] laser in 1961) that they began to be used in medical applications. In 1967, Endre Mester in Hungary noticed the ability of the HeNe laser to increase hair growth  and stimulate wound healing in mice , and, shortly afterward, he began to use lasers to treat patients with nonhealing skin ulcers . Since those early days, the use of low-power lasers (as opposed to high-power lasers that can destroy tissue by a photothermal effect) has steadily increased in diverse areas of medical practice that require healing, prevention of tissue death, pain relief, reduction of inflammation, and regenerative medicine. Some of the different organ systems, diseases, and injuries that have been effectively treated with low-level laser therapy (LLLT) are schematically shown in Figure 1.
Nevertheless, this modality, which is variously known as LLLT or photobiomodulation, remains controversial. The reasons for this lack of general acceptance among both the medical community and the general public at large are 2-fold. First, widespread uncertainty and confusion exists about the mechanisms of action of LLLT at the molecular, cellular, and tissue levels. Second, a large number of parameters (eg, wavelength, fluence, irradiance, treatment timing and repetition, pulsing, and polarization) can be chosen in designing LLLT protocols. Furthermore, a biphasic dose response exists in laser therapy , which describes the observation that increasing the overall “dose” of the laser either by increasing the power density or by increasing the illumination time may have a counter-productive effect compared with the benefit obtained with lower doses. Taken together, these considerations may explain why a number of negative studies have been published; however, this should not be taken to imply that LLLT in general does not work but rather that the laser parameters used in those particular studies were ineffective.
In recent years, the development of light-emitting diodes (LEDs) as alternative light sources for LLLT has added to the confusion. These devices produce light with wavelengths similar to those of lasers, but they have broader output peaks (ie, they are less monochromatic) and lack the coherence that is a particular feature of laser light. LEDs have the advantage of being significantly less expensive than laser diodes (by a factor of approximately 100 on a milliwatt basis), and the LLLT community is engaged in a vigorous ongoing debate about their respective benefits.
This review covers the mechanisms that are thought to operate at molecular and cellular levels in LLLT. Many of the most compelling applications of LLLT are in the field of neurology (both central and peripheral). Many serious brain diseases and injuries can be successfully treated with noninvasive transcranial laser therapy. Furthermore, in the peripheral nervous system, LLLT can be used effectively for nerve regeneration and pain relief.
LLLT uses low-powered laser light in the range of 1-1000 mW, at wavelengths from 632-1064 nm, to stimulate a biological response. These lasers emit no heat, sound, or vibration. Instead of generating a thermal effect, LLLT acts by inducing a photochemical reaction in the cell, a process referred to as biostimulation or photobiomodulation. Photo-biology works on the principle that, when light hits certain molecules called chromophores, the photon energy causes electrons to be excited and jump from low-energy orbits to higher-energy orbits. In nature, this stored energy can be used by the system to perform various cellular tasks, such as photosynthesis and photomorphogenesis. Numerous examples of chromophores exist in nature, such as chlorophyll in plants, bacteriochlorophyll in blue-green algae, flavoproteins, and hemoglobin found in red blood cells. The respective colors of chromophores are determined by the part of the spectrum of light they absorb: chlorophyll is green, flavoprotein is yellow, and hemoglobin is red .
Mitochondria are considered the power generators of the eukaryotic cell, converting oxygen and nutrients through the oxidative phosphorylation process and electron transport chain into adenosine triphosphate (ATP), as shown in Figure 2. The basic idea behind cellular respiration is that high-energy electrons are passed from electron carriers, such as reduced nicotinamide adenine dinucleotide (NADH) and the reduced form of flavin adenine dinucleotide (FADH2), through a series of transmembrane complexes (including cytochrome c oxidase [CCO]) to the final electron acceptor, generating a proton gradient. The gradient is used by FOF1 ATP synthase to produce ATP. Various in vitro experiments, such as those that use rat liver isolates, found that cellular respiration was upregulated when mitochondria were exposed to an HeNe laser or other forms of illumination. Laser irradiation caused an increase in mitochondrial products (such as ATP , NADH, protein, ribonucleic acid [RNA] ) and a reciprocal augmentation in oxygen consumption. A similar effect is produced when tissue that contains mitochondria is exposed to low-level radiation. Visible and near-infrared (NIR) light is absorbed by the organelle, and an upregulation of cellular respiration is observed .
Once it was observed that LLLT's mechanism of action is at the level of the mitochondria, it remained to be determined what specific structure within the mitochondria acted as the chromophore. Four membrane-bound complexes have been identified in mitochondria, each constituting an extremely complex transmembrane structure embedded in the inner membrane. Complex IV, also known as CCO, is a large transmembrane protein complex found in mitochondria, which is a component of the respiratory electron transport chain (Figure 3). CCO appears to absorb the same spectrum of light as that observed for the action spectra for the biological response to light in the NIR range. Thus it is reasonable to assume that CCO acts as an important chromophore in LLLT . CCO consists of 2 copper centers and 2 heme-iron centers that are capable of absorbing light over a wide range, including NIR.
The next reasonable question to consider is: What action does CCO modulate once it absorbs the energy from light? On the cellular level, LLLT may cause photodissociation of nitric oxide (NO) from CCO. In a stressed cell, NO produced by mitochondrial NO synthase displaces oxygen from CCO, which results in a downregulation of cellular respiration and a subsequent decrease in the production of energy-storing compounds, such as ATP. By dissociating NO from CCO, LLLT prevents the displacement of oxygen from CCO and thereby promotes unhindered cellular respiration  (see Figure 4). Increased CCO enzyme activity can be measured ; increased ATP production  and increased electron transport  also have been reported. The basic idea behind cellular respiration is that high-energy electrons are passed from electron carriers, such as NADH and FADH2, through a series of transmembrane complexes (including CCO) to the final electron acceptor. Increased cellular ATP produced by LLLT may contribute to the positive effects, both by raising cellular energy levels and by upregulating the cyclic AMP molecule (biochemically formed from ATP) that is involved in many signaling pathways.
Oxygen acts as the final electron acceptor and is, in the process, converted to water. Part of the oxygen that is metabolized produces reactive oxygen species (ROS) as a natural by-product. ROS (eg, superoxide and hydrogen peroxide) are chemically active molecules that play an important role in cell signaling, regulation of cell cycle progression, enzyme activation, and nucleic acid and protein synthesis . Because LLLT promotes the metabolism of oxygen, it also acts to increase ROS production. In turn, ROS activates certain redox-sensitive transcription factors such as nuclear factor-κB [NF-κB] and activator protein 1, which leads to the upregulation of various stimulatory and protective genes. The ultimate effect of LLLT is likely to be produced by transcription factor activation, which modulates the host's downstream cellular and tissue responses (see Figure 5).
Almost certainly, other mechanisms through which LLLT produces its effects are at play in addition to the one just described. For example, NO is a potent vasodilator via its effect on cyclic guanine monophosphate production. Cyclic guanine monophosphate is also involved in many other signaling pathways. LLLT may cause the photodissociation of NO from intracellular stores (ie, nitrosylated forms of both hemoglobin and myoglobin, in addition to CCO) . LLLT promotes the synthesis of deoxyribonucleic acid (DNA) and RNA  and increases the production of proteins . It also modulates enzymatic activity , affects intracellular and extracellular pH [17,18], and accelerates cell metabolism [18,19]. The expression of multiple genes related to cellular proliferation, migration, and the production of cytokines and growth factors also have been shown to be stimulated by low-level light .
Light is a powerful force and has a myriad of effects. The specific mechanisms of action may vary among various clinical applications of LLL and will be discussed in the respective sections below. Furthermore, in spite of a great number of studies that explored how LLLT works, the exact mechanism of action remains to be fully elucidated.
Transcranial LLLT (808 nm) has significantly improved recovery after ischemic stroke in rats when they received one treatment 24 hours after sustaining a stroke [21,22]. Stroke was induced in rats by 2 different methods: (1) permanent occlusion of the middle cerebral artery through a craniotomy or (2) insertion of a filament. The laser was used transcranially on the exposed (shaved skin) skull by placing the tip of the 4-mm diameter fiber optic onto the skin at 2 locations on the head (3 mm dorsal to the eye and 2 mm anterior to the ear) on the contralateral hemisphere to the stroke. These locations had been determined from prior measurements to be sufficient to illuminate 1 brain hemisphere as a result of dispersion of the laser beam by the skin and the skull. Results of previous studies had shown that LLLT of the contralateral, or both hemispheres, demonstrated no difference in functional outcome . An NIR gallium arsenic diode laser was used transcranially to illuminate the hemisphere contralateral to the stroke at a power density of 7.5 mW/cm2 to the brain tissue . In both models of stroke, the neurologic deficits at 3 weeks after stroke were significantly reduced (by 32%) (P < .01) in the laser-treated rats compared with control subjects.
In this study, the number of newly formed neuronal cells, assessed by double immunoreactivity to bromodeoxyuridine and tubulin isotype III, as well as migrating cells (double Cortin immunoreactivity), was significantly elevated in the subventricular zone of the hemisphere ipsilateral to the induction of stroke when treated by LLLT [21,22]. No significant difference in the stroke lesion area was found between control and laser-irradiated rats. The researchers suggested that an underlying mechanism for the functional benefit after LLLT in this study was possible induction of neurogenesis. Results of other studies also suggested that, because improvement in neurologic outcome may not be evident for 2-4 weeks in the poststroke rat model, delayed benefits may in part be due to induction of neurogenesis and migration of neurons [24,25]. In addition, transcranial LLLT may prevent apoptosis and improve outcomes by exerting a neuroprotective effect, although these exact mechanisms are poorly understood .
Other studies in rat and rabbit models also have observed that transcranial LLLT improves functional outcome after stroke [25,27,28]. A recent rabbit study combined transcranial LLLT with thrombolytic therapy by using tissue plasminogen activator, with no increase in bleeding and good safety .
In the aforementioned studies, it has long been hypothesized that increased mitochondrial function (ie, increased ATP production) in brain cells irradiated with NIR LLLT was one of the major mechanisms involved with the beneficial behavioral effects observed after LLLT treatment. A recent animal study with rabbits has shown a direct relationship between the level of cortical fluence (energy density) delivered (in J/cm2) and cortical ATP content in embolized rabbits . Five minutes after embolization (right carotid), the rabbits were exposed to 2 minutes of NIR transcranial LLLT with use of an 808-nm laser source (continuous wave [CW] or pulsed wave [PW] at 100 Hz or at 1000 Hz on the skin surface, posterior to bregma at midline). Three hours after embolization, the cerebral cortex was excised and processed for measurement of ATP content. Embolization decreased cortical ATP content in ischemic cortex by 45% compared with naive rabbits.A linear relationship up to 4.5 J/cm2 in fluence delivered, was observed for the relationship between cortical fluence (in J/cm2) verus percent increase in cortical ATP content (over sham-treated embolized rabbits). This linear relationship was observed with a power density of 7.5 mW/cm2 CW (0.9 J/cm2), where an increase of 41% in cortical ATP was observed; and with a power density of 37.5 mW/cm2 PW (100 Hz, 4.5 J/cm2), where an increase of 157% in cortical ATP was observed. An increase in cortical ATP of 221% was observed with fluence of 31.5 J/cm2, delivered with a power density of 262.5 mW/cm2 PW, 1000 Hz. This suggests that a near-plateau effect was present regarding the fluence level delivered above 4.5 J/cm2. It was surprising, however, that the increased cortical ATP levels of 157% and 221%, were higher than those measured in naive rabbits that had never suffered stroke. Because the authors observed that the PW modes (100 Hz and 1000 Hz) were more effective than the CW mode to increase cortical ATP, they hypothesized that in future stroke studies in animals and in humans, even greater improvement in clinical rating scores might be achieved by optimizing the method of NIR transcranial LLLT delivery, including the length of treatment and the mode of treatment (PW).
Transcranial LLLT has been shown to significantly improve outcome in acute human stroke patients when applied approximately 18 hours after the stroke occurs over the entire surface of the head (20 points in the 10/20 electroencephalographic system), regardless of the stroke location . Only one LLLT treatment was administered, and, 5 days later, significantly greater improvement was found in the real-treated group but not in the sham-treated group (P < .05, National Institutes of Health Stroke Severity Scale). This significantly greater improvement was still present 90 days after –the stroke occurred, at which time 70% of the patients treated with real LLLT had a successful outcome compared with only 51% of control subjects. An NIR (808 nm) laser was used, which delivered a fluence of 0.9 J/cm2 over the entire surface (2 minutes per each of the 20 points; power density of 7.5 mW/cm2).
In a second, similar study with the same transcranial LLLT protocol, an additional 658 acute stroke patients were randomly assigned to receive real or sham treatments of transcranial LLLT. Similar significant beneficial results (P < .04) were observed for the patients who had a moderate or moderate to severe stroke (n = 434) and received the real laser protocol but not for the patients who had a severe stroke . When all 656 cases were included in the data analysis (including the severe stroke cases), no significant real versus sham LLLT effect was seen. When data for both stroke studies were pooled (n = 778 [120 plus 658]) [31,32], a highly significant beneficial effect was seen for the real transcranial LLLT group (P = .003) compared with those who received the sham laser treatment .
Lampl et al  wrote that “Although the mechanism of action of infrared laser therapy for stroke is not completely understood . . . infrared laser therapy is a physical process that can produce biochemical changes at the tissue level. The putative mechanism . . . involves stimulation of ATP formation by mitochondria and may also involve prevention of apoptosis in the ischemic penumbra and enhancement of neurorecovery mechanisms.”
To date, no studies have been conducted to examine transcranial LLLT treatment of chronic stroke patients. Naeser et al  studied the application of LLLT-laser acupuncture (instead of needles) to stimulate acupuncture points on the body in chronic stroke patients with paralysis. Seven stroke patients (range, 48-71 years; 5 men) were treated, 5 of whom had single left hemisphere stroke, and 2 of whom had single right hemisphere stroke. Five patients were treated for hemiplegia, including severely reduced or no voluntary isolated finger movement, and 2 patients had hand paresis only. Six of the 7 patients received laser acupuncture during the chronic phase after the stroke had occurred (10 months to 6.5 years after stroke onset), clearly beyond the spontaneous recovery phase, which is considered to be up to 6 months after the stroke occurs [35,36]. The patients served as their own controls; no sham LLLT was administered. One patient (who had hand paresis) received LLLT during the acute phase after the stroke occurred (1 month after the stroke occurred). The patients did not receive any physical therapy or occupational therapy treatments while participating in this study.
A 20-mW gallium aluminum arsenide (780 nm) NIR CW laser with a 1-mm-diameter aperture was used (Unilaser, Copenhagen, Denmark). (At the time of this study, more powerful red or NIR lasers were not yet available.) Treatment consisted of stimulation of shallow acupuncture points (located on the hands and face) for 20 seconds per point (51 J/cm2). Deeper acupuncture points (located on the arms and legs) were treated for 40 seconds per point (103 J/cm2). Acupuncture points were treated on both the paralyzed side (arm, leg, and/or face) and on the nonparalyzed side by using primarily acupuncture meridians of the large intestine, triple warmer, gall bladder, liver, small intestine, and stomach . The patients were treated 2-3 times per week for 3-4 months. They received a total of 20, 40, or 60 treatments (based on patient availability and transportation). Within a few days before the first treatment and a few days after the last treatment, physical therapy and/or occupational therapy testing was performed by therapists blinded to the acupuncture treatment program to which the patient had been assigned: LLLT, real or sham needle, or no acupuncture. Overall, 5 of 7 of the patients (71.4%) showed improvement.
The 2 patients who showed no improvement had severe paralysis. We have observed that severity of paralysis and potential for improvement after LLLT-laser acupuncture (or needle acupuncture) is related to lesion location on chronic computed tomography (CT) scan acquired at least 3 months poststroke onset. Patients with lesion in more than half of the “periventricular white matter area” (PVWM) (adjacent to the body of the lateral ventricle, superior to the posterior limb, internal capsule), an area containing multiple efferent and afferent pathways (eg, thalamocortical, occipitofrontal, pathways from SMA/cingulate gyrus to the body of caudate, medial subcallosal fasciculus, and others), had severe paralysis which did not improve following LLLT-laser acupuncture (or needle) acupuncture treatments [34,37,38]. This area is diagrammed in Figure 6. The CT scan for a chronic stroke patient who had good response after LLLT-laser acupuncture treatments [34,37,38]. This area is diagrammed in Figure 7.
The 3 chronic stroke patients with hemiplegia who showed improvement after LLLT had an increase of 11%-28% in isolated, active range of motion for shoulder abduction, knee flexion, and/or knee extension (mean, 15.8%; SD, 7.1). This percentage increase after LLLT-laser acupuncture was similar to that observed after a series of 20 or 40 needle acupuncture treatments [37,38]. The person with hand paresis who was treated with LLLT at 33 months after stroke onset showed an increase of 2-6 lb in grip strength, 3-jaw chuck, tip pinch, and lateral pinch in the affected hand. These results are similar to those obtained with needle acupuncture . These findings are intriguing and suggest that some recovery of motor function can occur with needle acupuncture or LLLT acupuncture applied to body acupuncture points in chronic stroke patients.
A reduction in hand spasticity also has been observed when chronic stroke patients are treated with a combination of red-beam laser applied to hand acupuncture points plus microamps transcutaneous electrical nerve stimulation (TENS). Figure 8 shows an immediate reduction in hand spasticity after the first hand treatment when LLLT-laser acupuncture and microamps TENS were used with 2 chronic stroke patients. This LLLT and microamps TENS hand treatment program also may be used with patients who have hand spasticity related to other etiologies, including, for example, traumatic brain injury (TBI), “stiff man syndrome,” and spinal cord injury (SCI) (personal observation, M.A.N., 2001). Similar to red and NIR LLLT, microamps TENS increases ATP levels when applied to the skin . However, Cheng et al  observed that when stronger milliamps TENS was used (eg, similar to conventional TENS), the ATP levels were decreased. Hence when microamps TENS is used (as shown in Figure 8) , it is advisable to keep the sensation below threshold for the patient to increase ATP (not decrease ATP).
Each year in the United States, more than 1.4 million new cases of TBI occur, and more than 80,000 persons are left with permanent disability . Mild TBI (mTBI) from single and multiple events is the most frequent type of head injury experienced by military personnel deployed to Iraq and Afghanistan . TBI is known to cause damage that ranges from observable to microscopic throughout the gray and white matter of the brain. Diffuse axonal injury  is often observed in the anterior corona radiata and frontotemporal regions . Two regions highly susceptible to damage within the frontal lobes are the prefrontal cortex and the anterior cingulate gyrus. Cognitive processing problems result from tissue damage and inefficient cellular function in these brain regions. The prefrontal cortex is involved with maintaining, monitoring, and manipulating information in working memory  and particularly in sustained attention [47,48].
In the first reported study of the use of transcranial LLLT to treat traumatic brain injury, an animal model was used . Mice were subjected to closed-head injury (CHI) by using a weight-drop procedure, and 4 hours after CHI, either sham or real NIR LLLT (808 nm) was administered transcranially. The control group received no laser therapy (n = 8); the laser-treated group (n = 16) received 1 transcranial LLLT treatment by using a 200-mW, 808-nm NIR laser with a 3-mm-diameter probe tip (Photothera Inc, Carlsbad, CA). Either 10 or 20 mW/cm2 was administered. A single point was treated on the skull (a skin incision was made) that was located 4 mm caudal to the coronal suture line on the midline. The point was treated for 2 minutes (1.2-2.4 J/cm2). At 24 and 48 hours after CHI, no significant difference in motor behavior was seen between mice in the laser-treated and control groups. After 5 days, the motor behavior was significantly better (P < .05) in the laser-treated group; in addition, the neurobehavioral scores were 26%-27% better (lower scores indicated better motor behavior). At 28 days after CHI, the brain-tissue volume was examined for mice in each group. The mean lesion size of 1.4% in the laser-treated group (SD 0.1) was significantly smaller (P < .001) than in the control group (12.1%, SD 1.3). No difference in lesion size or behavior was observed in the mice treated with 10 mW/cm2 and those treated with 20 mW/cm2. The researchers suggested various possible mechanisms, including an increase in ATP, total antioxidants, angiogenesis, neurogenesis, heat shock proteins content, and an antiapoptotic effect, similar to observations reported after LLLT treatment of ischemic heart skeletal muscles [50-54].
Moreira et al  conducted a study in 2009 using phototherapy with low-intensity lasers and observed the effect on local and systemic immunomodulation after cryogenic brain injury in rats. Brain and blood samples were analyzed by enzyme-linked immunosorbent assay for the production of cytokines interleukin (IL)-6 , IL-10, IL-1b, and tumor necrosis factor (TNF)-α. The study concluded that laser phototherapy could positively affect the balance of IL-1b, TNF-α, and IL-6 in rats and thereby prevent cell death after TBI.
Wu et al  reported another mouse study of LLLT mediated by transcranial laser therapy. A nonfocal (diffuse) TBI was produced by a CHI caused by a calibrated weight-drop device. A neurologic severity score for each mouse was determined based on 10 standardized performance tests (involving beam balancing and maze exiting) administered at specified times. Mice with a neurologic severity score of 7-8 (moderately severe brain injury) were used in the study. Mice were given a single treatment to the top of the head with 36 J/cm2 of a 665-nm, 810-nm, or 980-nm laser 4 hours after the closed head TBI. Both 665-nm and 810-nm lasers were highly effective in improving the neurologic performance of the mice during the succeeding 4 weeks. The 980-nm wavelength was ineffective (negative control). We believe that this difference in results can be explained by the absorption spectrum of the different chromophores; CCO has peaks at 660 nm and 810 nm, whereas water has a peak at 980 nm.
In humans, 2 persons with chronic mTBI recently have been reported to have improved cognition after a series of treatments with transcranial, red, and NIR LEDs [57,58]. The LED cluster heads were applied to the forehead and scalp areas (the hair was not shaved off but was parted underneath each 2-inch-diameter LED cluster head). Each cluster head had 61 diodes (9 red 633-nm diodes and 52 NIR 870-nm diodes). Each diode was 12-15 mW, and the total power output was 500 mW. The LED cluster heads were applied to bilateral frontal, parietal, and temporal areas and to the mid-sagittal suture line.
Each LED cluster head was applied for 10 minutes per placement. With the device used here (parameters described above), 1 joule per cm2 (J/cm2) energy density was produced during every 45 seconds of exposure time. The energy density dose at the forehead-scalp was 13.3 J/ cm2; the power density was 22.2 mW/cm2 (±20%). The power density refers to the mW of power applied per cm2. The ± refers to the range of fluctuation (plus or minus 20%) on the power density per cm2. This power density is well below that used in other transcranial laser or LED studies to treat acute stroke cases or severe depression cases (225 mW/cm2) . It is estimated that only approximately 3% of the photons delivered to the forehead-scalp surface will reach 1 cm, to the cortex . The dose of 13.3 J/cm2 per placement area was estimated to deliver only 0.4 J/cm2 to the brain cortex. No sensation of heat or pain was reported during the LED application to the skin or scalp. These LED cluster heads (MedX Health Corp, Mississauga, Ontario, Canada) are approved by the U.S. Food and Drug Administration for treatment of musculoskeletal pain; they were used off-label for treatment of cognition in the mTBI cases. No potential existed for ocular damage because the LEDs produce noncoherent light. These LED cluster heads also have been approved by the Food and Drug Administration for home treatment.
A 66-year-old woman (case 1) began transcranial LED treatments 7 years after a motor vehicle–related TBI. Before LED treatment, she could focus on her computer for only 20 minutes. After 8 weekly LED treatments, her focused computer time increased to 3 hours. She has treated herself nightly at home for 5.5 years, with transcranial LED. She maintains her improved cognition at age 72 years.
Case 2 involved a 52-year-old retired, high-ranking female military officer who had a history of multiple TBIs. Her brain MRI showed frontoparietal atrophy. She was medically disabled for 5 months before beginning nightly transcranial LED treatments at home (see Figure 9, A and B). After 4 months of nightly LED treatments, she returned to work full time as an executive consultant for an international technology consulting firm and discontinued medical disability. Neuropsychological tests performed after 9 months of transcranial LED showed significant improvement in cognition (see Figure 9, C). After LED treatments, she improved on tests of executive function (inhibition and inhibition accuracy, +2 SD) and on memory (immediate and delayed recall +1, +2 SD). The improvement of +1 or +2 standard deviations on her scores refers to the degree of improvement on her scores after 9 months of LED treatments (versus before LED treatments). The SDs are provided with the test materials, and they are based on the published norms for each test.
Both patients with TBI reported that they needed to continue with home treatments. If they stop treatment for 1 or 2 weeks, then their cognitive problems started to return. Both patients with TBI reported improved sleep. The second patient with TBI reported a decrease in her posttraumatic stress disorder symptoms after a few months of using the transcranial LEDs, and Schiffer et al  also reported a reduction in posttraumatic stress disorder symptoms in 3 of 10 patients with major depression who were treated with transcranial LED.
Several possible mechanisms may be associated with the improved cognition in the mTBI cases treated with transcranial LEDs . Mitochondria display a significant amount of dysfunction after TBI [61-63]. The primary mechanism for improvement posited in one study with human acute stroke patients was an increase in ATP, with photons being used by CCO in the mitochondria to increase ATP, especially in the cortex .
An increase in ATP after red and/or NIR LED treatments in patients with chronic TBI would have beneficial effects, including an increase in cellular respiration and oxygenation. Oxidative stress plays a role in the damage present after TBI . One hypothesis is that LLLT produces low levels of ROS in mitochondria of illuminated cells and that these ROS cause NF-κB activation via the redox sensitive sensor enzyme protein kinase D1, which results in upregulation of the mitochondrial superoxide dismutase . A single exposure of LLLT-LED in vitro with fibroblasts has been observed to increase NF-κB in the short term . In stimulated dendritic cells in the longer term, however, NF-κB and pro-inflammatory cytokines were reduced . Thus, in the long term, repeated LED treatments are hypothesized to decrease inflammation (less NF-κB) and upregulate gene products that are cytoprotective, such as superoxide dismutase, glutathione peroxidase, and heat shock protein 70 [54,69]. It is hypothesized that an overall protective response occurs with repeated LED treatments and that major ROS-mediated damage and chronic inflammation that occur in the brain after TBI may actually be reduced.
Acupuncture points located on the scalp were treated with the red-NIR LEDs . This includes points along the Governing Vessel (GV) acupuncture meridian, located on the midline of the skull (including, in part, the mid-sagittal suture line). Some acupuncture points located on the GV meridian have been used historically to help treat patients in coma  and stroke , for example, GV 16 (inferior to occipital protuberance), GV 20 (vertex), and GV 24 (near center-front hairline); these points were treated in both patients with TBI reported in this study.
Transcranial red-NIR LED may have irradiated the blood via the valveless, emissary veins located on the scalp surface but interconnecting with veins in the superior sagittal sinus (M. Dyson, oral personal communication, June 2009). If red-NIR photons penetrate deeply enough to reach the cortex, then it also is possible they are entering small vessels located between the arachnoid and the pia mater, including those that supply arterial blood to superficial areas of the cortex. Direct in vitro blood irradiation with a red-beam laser has been observed to improve erythrocyte deformability (flexibility) and rheology [72,73]. A beneficial effect from direct-laser blood irradiation in vivo has been observed during stenting procedures where a low-level, red-beam laser (10 mW, 650 nm) was used, with the beam placed directly into a coronary artery . The restenosis rate was reduced and no adverse effects or complications were noted. Thus blood irradiation at the scalp may have affected local intracerebral blood and circulation; however; whether this effect occurred is unknown and would require further study.
An increase in regional cerebral blood flow may have occurred, specifically to the frontal lobes. The second TBI case showed significant improvement on objective, neuro-psychological testing for executive function (inhibition) after administration of LED. These results suggest improved function in the prefrontal cortex and anterior cingulate gyrus regions. Significant improvement on “inhibition” on the Stroop test particularly suggests improved function of the medial prefrontal cortex, anterior cingulate gyrus area . It is possible that this medial prefrontal cortex area could have been treated with NIR photons, especially when the LED cluster head was placed over the midline, front hairline area. The dorsolateral prefrontal cortex also was likely irradiated when the LEDs were placed on the left and right high-frontal areas of the scalp. Increased regional cerebral blood flow also could have occurred in frontal pole areas with the TBI cases, as was observed in the recent transcranial LED study to treat major depression . Additional controlled studies with real and sham transcranial LLLT and LED are recommended to investigate whether these methods can be applied to improve cognition and reduce symptom severity in persons with acute and chronic TBI. The LED technology is not expensive ($1400 for a single LED cluster head and approximately $4000 to $5000 for a unit with 3 LED cluster heads). The transcranial LED treatment protocol can be used in the home.
The positive effects of transcranial laser therapy on stroke and TBI have led to early investigations into whether LLLT may have benefits for persons with degenerative brain disorders, which are a rapidly growing affliction of the world's aging population. Moges et al  tested whether LLLT had a role to play in treating familial amyotrophic lateral sclerosis (FALS), which is a neurodegenerative disease characterized by progressive loss of motor neurons and death. Mitochondrial dysfunction and oxidative stress play an important role in motor neuron loss in ALS. The study combined LLLT (with use of an 810-nm diode laser with 140-mW output power targeting a 1.4-cm2 spot area for 120 seconds using 12 J/cm2 energy density) and riboflavin to test the survival of motor neurons in a mouse model of FALS. Motor function (determined with use of the Rota rod test) was significantly improved in the LLLT group in the early stage of the disease. Immunohistochemical expression of the astrocyte marker glial fibrillary acidic protein was significantly reduced in the cervical and lumbar enlargements of the spinal cord as a result of LLLT.
Trimmer et al  carried out preliminary studies that may have relevance to Parkinson disease (PD). Mitochondria supply the ATP needed to support axonal transport, which contributes to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functi
This video shows before and after treatment footage of a patients with advanced Parkinsons. Treatment lasts about 2 weeks. Dr Riner is using the brain and neurostim setting on the brain, C5 Nerve Root and the Ulnar nerve in the elbow.
The TheraLazr is the prototype for the Avant LZ30 series of lasers.
video length: (2:03)
Objective: Set within the context of the 2015 International Year of Light and Light-Based Technologies,and of a growing and aging world population with ever-rising healthcare needs, this perspective and mini-review focuses on photobiomodulation (PBM) therapy as an emerging, cost-effective, treatment option for cancer (i.e., solid tumors) and other complex diseases, particularly, of the eye (e.g., age-related macular degeneration, diabetic retinopathy, glaucoma, retinitis pigmentosa) and the central nervous system (e.g., Alzheimer's and Parkinson's disease). Background data: Over the last decades, primary and secondary mechanisms of PBM have been revealed. These include oxygen-dependent and oxygen-independent structural and functional action pathways. Signal and target characteristics determine biological outcome, which is optimal (or even positive) only within a given set of parameters. Methods: This study was a perspective and nonsystematic literature mini-review. Results: Studies support what we describe as a paradigm shift or “quantum leap” in the understanding and use of light and its interaction with water and other relevant photo-cceptors to restore physiologic function. Conclusions: Based on existing evidence, it is argued that PBM therapy can raise the standard of care and improve the quality of life of patients for a fraction of the cost of many current approaches. PBM therapy can, therefore,benefit large, vulnerable population groups, including the elderly and the poor, whilehaving a major impact on medical practice and public finances.
The United Nations declared 2015 to be the International Year of Light and Light-Based Technologies (IYL 2015) in recognition of the vital role of light-based systems in our daily lives, and their growing importance to meeting the world's challenges in areas as diverse as energy, education, telecommunication, agriculture, and health.1 Although our perception of light is often limited to the visible band of the electromagnetic (EM) spectrum,2 both lower and shorter wavelengths are increasingly used in new medical technologies3 including soft, injectable, and bioresorbable electronics.4 Described as an imperative cross-cutting discipline of in the twenty-first century, light science has already revolutionized the physical sciences and industry. The control of light at the nanoscale has unveiled a plethora of phenomena, leading to powerful new applications and setting high expectations for years to come.5 In particular, light's ability to control materials and transport coded signals forms the bases for many new photonic devices and systems, wherein photons act as tailor-made EM energy packets that can perform various functions.
Here, we describe a paradigm shift or “quantum leap” in the understanding and use of light and its interaction with water and other relevant photoacceptors to control biologic function in medicine through photobiomodulation (PBM) therapy. We propose that progress will lead to the imminent inception of PBM therapy as a mainstream treatment for multiple complex diseases, including solid tumors, as well as neurodegenerative diseases (NDs) of the eye and central nervous system (CNS)6–10 (Fig. 1). PBM therapy can raise the standard of care and improve the quality of life of patients at a fraction of the cost of many current approaches. Thus, a “quantum leap” in PBM therapy will benefit large and vulnerable population groups, including the elderly and the poor, while having a major impact on medical practice and public finances.11 This is particularly important because the high price of drug therapies, which can reach hundreds of thousands of dollars per year,12 as well as a growing and aging world population, are putting a severe strain on family and public finances around the world.13
Flow chart illustrating fields of light-based technologies, highlighting photobiomodulation (PBM) therapy applied to complex diseases as a quantum leap in medical therapeutics.
Origin, Trajectory and Myriad Relationships in PBM's “Quantum Leap” in Medicine
Concurrent with progress in PBM therapy, a long history of discoveries has put medicine at the brink of a revolution in the use of light–water interactions for the treatment of complex diseases.7,8,10,14 Long ago, Albert Szent-Gyorgyi postulated that water was at the core of energy transfer in biological systems (i.e., quantum biology), and that that explained how energy from biomolecules could be translated into free energy for cells.15–17 Ling further elaborated on the physical state of water in living cells,18 and proposed on theoretical grounds that ordered layers of water could extend infinitely under ideal conditions.19,20Later, Huber proposed a structural basis of light energy and electron transfer in biology.21 More recently, Zewail and others showed that, with rapid laser techniques, it is possible to “see” how atoms in a molecule move during a chemical reaction.22 Light science has now reached microscales at the limit of recordable physical observation (e.g., resonant intermolecular transfer of vibrational energy in water at −100 fs)23,24showing, for example, the memory of persistent correlations in water structures within 50 fs, which is important in stabilizing biological systems.25 These and other tremendous achievements have changed our view of water, from a merely passive medium to an integral active player in the physiology of life, and have opened the gates to both direct measurement and control of physiological processes via light–water interaction.
State of the Art in PBM
In 2016, PBM therapy will be added to the MeSH database as an entry term for records spanning five decades of research.26 As argued by Anders et al., this is a key step, as it distinguishes PBM therapy from light-based devices used for heating of tissues, such as near infrared (NIR) lamps or other applications that rely on thermal effects for all or part of their mechanisms of action.26 In contrast, PBM therapy employs low-level monochromatic or quasimonochromatic light, currently from visible blue (∼400 nm) to far-infrared (FIR ∼3200 nm), to induce nonthermal (≤0.01°C) photochemical and photophysical effects. Nonlinear processes through which PBM therapy can stimulate or inhibit; that is, modulate, physiological activity depend upon signal-to-noise rate and target cell/tissue parameters.27–29 Thus, signal and target characteristics determine biological outcome, which is optimal (or even positive) only within a narrow set of parameters.13
Over the last decades, primary and secondary mechanisms of PBM at the tissue, cellular, and molecular levels have been revealed. These include two major structural and functional action pathways. The first, or classic, action pathway relates to oxygen-dependent mechanisms operated by oxidation-reduction enzymes of the respiratory chain, particularly cytochrome c oxidase (CcO), which is partly responsible for light energy absorption and transfer to cells and tissues.30 This pathway is associated to cofactors, pigments, metals, and proteins that act as key redox centers within the body's bioenergetic rack mechanism described by Huber.21 Nitric oxide (NO), as a first-level player, also has an activation and modulation role in the oxygen-dependent pathway.31–33
The second, or oxygen-independent, action pathway centers on the vital role of water not only as the prevalent medium of life but as an active molecule, capable of absorbing radiant energy (e.g., IR light) and transporting/transducing it along extended biological surfaces, from bulk water to confined water in nanoscopic tissue and cell spaces. Light–water dynamics precede/coexist with the classic oxygen-dependent action pathway and complement and facilitate energy transfer for increased adenosine triphosphate (ATP) production.29,34,35 As a point of comparison, correlated internal electron- and proton-transfer reactions have been tracked in real time into the oxidized enzyme (CcO), revealing an overall real time of 3.46 ms.36 This relay is slower by several orders of magnitude than total energy transport through water dynamics from bulk liquid water to confined spaces.34
Oxygen-independent light–water interactions may further power and modulate molecular signaling pathways and gene transcription factors via multiple nonmetabolic pathways.10,35 For examle, the energy of the drive force wave of an infrared pulsed laser device (IPLD) used in our group's previous studies (NIR 0.27 eV) is within the range of the strength of hydrogen bonds,29,37 and the IPLD carrier wave oscillates at a frequency (3x 10e6 Hz) that enters in vibrational resonance with the rate of electron transfer through the DNA double helix.29,37 Theoretical evidence suggests that these wave properties promote the activation of open state dynamics,38,39 allowing the activation of complex chaotic dynamics as well as the regulation of DNA replication and transcription, because the existence of open states in one place of the chain can influence the dynamics of other distant open states.29,34,35 Resulting effects match reported reductions in the frequency of chromosome aberrations induced by that low-energy laser irradiation,40 as well as theoretical,38,39 experimental,27,28 and clinical studies.41–48 These and other oxygen-independent PBM effects are channeled through metabolic control levels to regulate the energy-dependent path from the genotype to the phenotype.49,50
Light–Water Interactions and the Quantum Leap in PBM
We propose that the key to understanding and controlling the biophysics and biochemistry of higher-order organisms stems from their dual aqueous and energy-dependent nature. Water represents ∼70% by mass of an adult human body, or nearly 99% of total molecules by number, given water's low molecular weight. In addition, high-order organisms, including humans, can be represented as complex electrochemical (semiconducting) systems that comprise a vast array of energy-sensitive materials and machinery, such as ion pumps (e.g., chemically driven electron pumping through molecular wires, such as the D pathway in CcO),34 molecular motors (e.g., ATP synthase and Brownian biomotors), transistors-capacitors (e.g., cell membrane), liquid crystals (e.g., membrane structure), and rechargeable electrolytic biological batteries (e.g., hydrophilic interface in cells/tissues). Life system's double nature, whose two main structural and functional pillars are energy and water joined to biomolecules, has, therefore, tremendous consequences for life and health.
Water's permittivity, calculated considering the system as a plane capacitor, is generally high. Therefore, radiant energy can penetrate and be absorbed by tissues to provide powerful tools in medicine.51 One example is the exclusion zone (EZ) described by Pollack.52 High-energy EZ water forms along hydrophilic surfaces (e.g., tissue interfaces) in response to radiant energy.53 Remarkably, EZ water can separate and store electrical charges, and can release up to 70% of such charges when it is perturbed, such as by injury-induced redox potentials.54 We have argued that supplied energy can power and modulate cellular work and signaling pathways, even when the metabolic energy pathway has been compromised, steering cells toward or away from programmed cell death.34 EZ water may, thus, act as an electrolytic bio-battery,35 which can efficiently and selectively transfer energy to sites expressing redox injury potentials, as found in cancer and other complex diseases, triggering reparative and regenerative mechanisms that can lead to restoring homeostasis/homeokinesis and, ultimately, health.29,34,35
Experimentally, IR energy absorption by water has been recently modeled in a porcine model, confirming that absorption depends upon fluence and wavelength. Further, the higher the concentration of water in tissues, the higher IR energy absorption will be.55 This is consistent with controlled clinical studies in solid tumors and complex ophthalmic and neurologic diseases,9,46,56 as well as molecular, biochemical, biophysical, and metabolic mechanistic support for a quantum leap in medical therapeutics based on the simple, but powerful, idea that properly tailored light can power and modulate physiologically reparative mechanisms.30,57–62
Cancer and Tumor Microenvironments
The bases of our understanding of cancer are constantly being questioned and revised, leading to new treatment goals. In a paradigm-changing editorial, Prendergast recently argued that “disorders in microenvironment and peripheral systems that control cancer might increasingly be viewed as primary rather than secondary factors in the root nature of cancer as a clinical disease.” This constitutes “a crucial and radical distinction from prevailing thought, since it implies that cancer may be a symptom of an underlying clinical disorder, rather than the root problem itself that needs to be addressed.” 6,63
Prendergast further suggested that “effective treatment of cancer may not necessarily entail understanding or addressing this complexity, but mastering the use of tissue or systemic systems that have the inherent ability to do so.” Hence, a common thread linking emerging perspectives in oncology and PBM therapy may well be the restitution of tissue homeostasis-homeokinesis via light-energy supplementation, a microenvironment effect that comprises and extends the Warburg effect previously discussed by our group.57,64–67
Photobiomodulation and Cancer
As far back as 1964–1966, McGuff et al. showed 64,65 that “laser energy has a selective effect on certain malignant tumors, resulting in their progressive regression and ultimate dissolution.” Following years of controversy,66,67 editorials by Karu68 and Lanzafame11,69 now stress evidence supporting the potential anticancer effects of PBM.11,68,69 New data confirm that PBM under certain parameters is safe for use in cancer patients.60 This is in accord with clinical results from our group using the abovementioned proof of concept IPLD.4,44
A phase I trial in patients with advanced neoplasias demonstrated that the IPLD studied was safe for clinical use and improved performance status and quality of life.41 Antitumor activity was observed in 88.23% of patients with 10 years of follow-up.41
In that series, T2-weighted MRI data showed increased water content of tumor heterogeneities42,44 preceding tumor-volume reduction and a therapeutic anticancer effect.42,44 Structural, kinetic, and thermodynamic implications of these changes in water dynamics have been analyzed at the tissue, cell, and interstitial levels.27 In conjunction, selective activation of programmed cellular death [i.e., apoptosis, necrosis, and anoikis (cell death by loss of cell adhesion)] and cytomorphologic modification (e.g., reduced size, increased roundness, increased vacuoles) were documented in neoplastic cells, but not in peripheral tissues.8,42 Modulation of cluster of differentiation (CD)4 CD45RA+, CD25 activated, tumor necrosis factor alpha (TNF-α), and soluble interleukin (IL)-2 receptor (sIL-2R) was further documented.43These hallmark results, supported by independent data,70–72 demonstrate that PBM therapy can modulate antitumor effects,6,8 in sharp contrast with long-held views.45,73,74 This evidence is also consistent with growing experimental and clinical reports from multiple other authors.60,75–82
PBM and Ophthalmic and Neurodegenerative Disorders
Recent evidence underscores common mechanisms between cancer and NDs of the eye and CNS. Research suggests that oxidative proteome damage may be the most likely cause of aging and age-related maladies such as cancer and other complex diseases, including NDs.83 Findings also show “common mechanisms of onset,” with a focus on genes such as DJ-1 and Myc-Modulator 1 (MM-1) and signaling pathways that contribute to the onset and pathogenesis of cancer and NDs such as retinitis pigmentosa (RP), Parkinson disease (PD), and cerebellar atrophy.”84 Finally, both disease groups are profoundly energetic in nature, featuring prominent deterioration of metabolic energy pathways.10
External light energy supplementation has been shown to generate neuroprotective, vasoprotective, baroprotective, immunomodulatory, and regenerative effects (Fig. 2). 47 We have documented that such effects may be activated and modulated locally and/or remotely via oxygen-dependent and oxygen-independent pathways that can encompass extended biologic surfaces and may even reach avascular eye tissues (i.e., cornea, lens, aqueous humor, and vitreous) noninvasively. Although a full elucidation of involved mechanisms escapes the scope of this perspective and mini-review, a very brief discussion of results from multiple authors is given subsequently.
Electromagnetic (light) energy supplementation based on water–light interactions. Upper left side shows classic oxygen (O2) dependent pathways by which light energy generates adenosine triphosphate (ATP)/ guanosine-5′-triphosphate (GTP) and other high-energy molecules. Upper right side shows O2 independent pathways by which photoinduced, nonlinear, oscillations in water provide energy for cellular work, signaling, and gene transcription. Top center shows interfacial exclusion zone (EZ) water, which acts as a selective rechargeable electrolytic bio-battery. Together, these pathways activate and modulate physiologically reparative mechanisms which, at appropriate irradiation parameters, can generate neuroprotective, vasoprotective, baroprotective, immunomodulator, and regenerative effects locally and remotely, promoting homeostasis/homeokinesis through the coupling and synchronization of biophysical, biochemical, biomechanical, and hydrodynamic oscillators, as guided by the second law of thermodynamics. Arrows point to the sequence and direction of events. (Updated from reference 47. Authors retained copyright.)
PBM has shown promise in the treatment of diabetic retinopathy (DR),85,86 age-related macular degeneration (AMD),46 glaucoma,47 RP,87 Stargardt disease,88 Leber's hereditary optic neuropathy,89 Alzheimer's disease (AD), and PD, 90,91 among other conditions.89 Strikingly, although each of these NDs has different etiologies and pathogeneses, “they frequently induce a set of cell signals that lead to well-established and similar morphological and functional changes, including programmed cell death. Furthermore, oxidative stress, activation of apoptotic pathways and inflammatory response, are common features in all these diseases.”92
Remarkably, PBM can modulate apoptosis as well as necrosis.42,45,47 PBM can also be both pro-oxidant in the short term, but antioxidant in the long term,93 thus modulating reactive oxygen species (ROS) generation. We also found clinical evidence of immune regulatory effects over inflammation during treatment of solid tumors with the IPLD, a NIR diode laser pulsed at a frequency of 3 MHz.43 These results are in agreement with the regulating role of the vagal reflex on the inflammatory reflex reported by Tracey, using an electronic device that stimulated nerves to treat inflammation.71,72
In addition, PBM has been shown to protect against retinal dysfunction and photoreceptor cell death in rodent models of retinal injury and retinal degeneration.94 PBM has been further reported to attenuate oxidative stress and inflammation in primary astrocytes induced by amyloid β peptide (Aβ),95 and to reduce Aβ-induced apoptosis,96 which is thought to play a major role in AD. Nevertheless, it has been argued that red to NIR light cannot be transmitted through the scalp to the brain more than a few centimeters,97 which makes it nearly impossible to noninvasively treat AD with PBM 98 using conventional (direct) delivery systems/methods. Similarly, although an absence of adverse effects from 670 and 830 nm PBM applied to the retina in Sprague Dawley albino rats has been reported,94 extreme care must be taken to avoid photodamage of the eye99 from direct PBM procedures.
Conversely, we published an interventional case report of a patient with bilateral geographic atrophic AMD (gaAMD) and associated neurologic disease treated noninvasively, indirectly, and at a distance (i.e., remotely) from ocular structures and the CNS with the above-referenced IPLD/photo-infrared pulsed bio-modulation (PIPBM).46 Results showed neurologic improvement, transitory color vision, enhanced visual acuity, full-field electroretinogram (ERG) modifications toward a normal rhythm, drusen mobilization, decreased lens opacity, and lower intraocular pressure (IOP), in accord with a retrospective noncomparative data analysis from the phase I trial of patients with advanced cancer treated with the IPLD,41 which showed statistically significant evidence of a therapeutic hypotensor effect over IOP,47 and they are consistent with the positive neurological evolution of two trial patients.
Moreover, although trial participants did not develop media opacity, one pre-existing incipient cataract in the right eye of a patient (transitional meningioma) became denser and slightly smaller 3 months post-treatment, and remained unchanged 1 year post-treatment. The left eye lens of the same patient was unaffected. Although the finding could be part of the natural history of the cataract, we stressed that possible deterministic effects related to the initial metabolic or biochemical state of lens opacities should be studied.41
In accordance with the what was described, a robust body of evidence suggests that protein misfolding, insolubility, and aggregation are at the root of both cataracts and other diseases including AD, PD, and Huntington's disease,100 and that external EM energy (light) supplementation can have reparative effect on protein misfolding, activating and modulating metabolic control levels of protein folding/unfolding.10,34In addition, PBM effects on targets such as heat shock proteins (α crystalline), enzymes of the antioxidative system, Na+-K+-ATPase, Ca +2-ATPase, aquaporins (AQPs), and ion pumps have been referred to as part of mechanisms that could have influenced the response observed in the lens on the cases studied.46 We further proposed that, among other effects, PBM can stimulate and/or substitute ATP production via water dynamics, which is vital for the activation and inactivation kinetics in phototransduction.46 PBM can also affect the synthesis of molecules in a liquid crystalline (LC) state (e.g., self-assembly of lipids, water, and other biomolecules such as proteins and sterols, which are sensitive to temperature and/or electric fields) If confirmed, the latter may have multidisciplinary applications in medicine and biology in areas such as photovision, in which LCs are essential functional components.28
A first rapid communication referring to the retina and optic nerve additionally showed first evidence of EZ water as a selective rechargeable bio-battery applicable to PBM, suggesting a new understanding of the eye's energetic environment, which may have deep implications in ocular physiology as well as in the pathophysiology, diagnosis, and treatment of blinding diseases using light-based therapies.48 Therefore, as a promising alternative to drug therapies,101 or in combination with other treatments, PBM therapy may be developed into a viable therapeutic approach with multidisciplinary applications in ophthalmology and neuroscience,46 inducing and modulating physiologically reparative and regenerative effects that can favor homeostasis/homeokinesis27–29 through the coupling and synchronization of biophysical, biochemical, biomechanical, and hydrodynamic oscillators, as guided by thermodynamics.
Treatment Costs and Availability
At the 2015 American Society of Clinical Oncology (ASCO) annual meeting, Dr. Leonard Saltz, chief of gastrointestinal oncology at Memorial Sloan Kettering Cancer Center, discussed the high cost of cancer drugs. He argued that “the unsustainably high prices of cancer drugs is a big problem, and it's our problem,” citing as examples the cost of nivolumab ($28.78/mg) and ipilimumab ($157.46/mg), which is “approximately 4000 times the cost of gold.”102 Previously, >100 oncologists had protested the high price of cancer drugs, also calling them economically “unsustainable.” They noted that, of 12 cancer drugs approved in 2012, 11 were priced > $100,000 per year,103 with multiple drugs often being required for extended periods. Such high prices and their impact on families, governments, and society at large are leading some to propose that cost should be considered a “financial toxicity” to be assessed with other toxicities when treatments are considered by doctors and patients.104 In contrast, although it has been estimated the cost of developing new drug therapies can run up to USD $1.3–$1.7 billion,105,106 the development cost of new photonics devices can be substantially lower, which can lower therapy costs and increase treatment availability. For the same reasons, PBM can also offer a noninvasive and cost-effective therapeutic option for patients with NDs of the retina, brain, and beyond.56, 85
The celebration of the IYL 2015 by the United Nations1 is a fitting time to announce what we describe here as a “quantum leap” in PBM therapy. It is also a good opportunity to ensure that policy makers and the medical community become aware of and embrace the immense potential of light-based medical technologies, especially PBM therapy, as an emerging treatment option for cancer and other complex diseases.107 Although not all tissues respond to PBM therapy,69 in vitro and in vivo xenografts and evidence from clinical studies does suggest that it is time to begin considering PBM therapy as a potential drug equivalent.11,108 In addition, PBM therapy may have minimal or no adverse effects, improve quality of life and functional status and raise the current standard of care for many cancer patients when used alone or in combination with other therapies.9 PBM therapy further represents a novel hope for the treatment of numerous eye and neurologic diseases. And as stated, PBM may be developed at a lower cost than many current treatments,8,10 which can help meet the healthcare needs of an increasing and aging world population. As such, this perspective and mini-review focuses on the large potential tangible contributions of light-based therapies for large demographic segments of the population, such as aging “baby boomers” who are expected to face a higher incidence of diseases such as cancer, AMD, DR, glaucoma, RP, AD, and PD, as well as other neurologic diseases in the next 15 years. In light of the growing costs of drugs and their impact on developed and developing countries, we propose that PBM therapy may offer a novel, safe, and effective therapy choice that would be more accessible to large vulnerable groups, such as the poor and the elderly.
Concurrently with the United Nations' declaration of 2015 as the year of light and light-based technologies, PBM therapy stands at the brink of delivering a new generation of treatments for complex diseases. New PBM therapies will preserve quality of life and raise standard of care in an efficient and cost-efficient manner. This will particularly benefit the most vulnerable demographic sectors, such as the elderly and the poor, and reduce the strain of growing healthcare costs in both industrialized and developing countries. We propose that such developments and their imminent impact represent a paradigm shift or “quantum leap” in PBM therapy and medicine at large.
We thank Jesús Alberto Santana-Rodríguez for reviewing and editing this article, and Luis Rafael Santana-Rodríguez for design and technical support. This study was supported by Fundalas, Foundation for Interdisciplinary Research and Development.
Author Disclosure Statement
No competing financial interests exist.
This book covers an astonishing amount of information in its near thousand pages, everthing from basic laser physics to dental, and veteranary useage. Here are some of its contents:
High-Tech Acupuncture with Laser Light
Use of photostimulation including low?level light emitting diode (LED) therapy has broadened greatly in recent years because it is compact, portable, and easy to use. Here, the effects of photostimulation by LED (610 nm) therapy on ischemic brain damage was investigated in mice in which treatment started after a stroke in a clinically relevant setting. The mice underwent LED therapy (20 min) twice a day for 3 days, commencing at 4 hours post?ischemia. LED therapy group generated a significantly smaller infarct size and improvements in neurological function based on neurologic test score. LED therapy profoundly reduced neuroinflammatory responses including neutrophil infiltration and microglia activation in the ischemic cortex. LED therapy also decreased cell death and attenuated the NLRP3 inflammasome, in accordance with down?regulation of pro?inflammatory cytokines IL?1β and IL?18 in the ischemic brain. Moreover, the mice with post?ischemic LED therapy showed suppressed TLR?2 levels, MAPK signaling and NF?kB activation. These findings suggest that by suppressing the inflammasome, LED therapy can attenuate neuroinflammatory responses and tissue damage following ischemic stroke. Therapeutic interventions targeting the inflammasome via photostimulation with LED may be a novel approach to ameliorate brain injury following ischemic stroke.
Effect of post?ischemic low?level light emitting diode therapy (LED?T) on infarct reduction was mediated by inflammasome suppression.
Ischemic stroke, a cerebrovascular insult, is the most common cause of physical disabilities worldwide. However, the only FDA approved treatment is tissue plasminogen activator that must be administered up to 4.5 hours after stroke onset 1. Therefore, identifying new stroke therapeutics would address a significant unmet medical need. Ischemic stroke initiates a complex cascade of events that leads to focal brain damage, and in which inflammation plays a significant role 2. The inflammatory response includes activation of resident microglia and production of pro?inflammatory cytokines 3, followed rapidly by infiltration and accumulation of neutrophils and monocytes/macrophages in microvessels and ischemic cerebral parenchyma 4.
Inflammatory mechanisms that contribute to cell death in cerebral ischemia are mediated by a multi?protein complex called the inflammasome 5-9. The key component is NOD?like receptor pyridine domain?containing (NLRP) protein, which initiates inflammasome activation when bound by its ligand. More specifically, the NLRP1 and NLRP3 inflammasomes are cytosolic complexes containing NLRP1 or NLRP3 receptor protein, ASC (apoptosis?associated speck?like protein containing a caspase recruitment domain), X?linked inhibitor of apoptosis (XIAP), precursor caspase?1 and/or precursor caspase?11 10. First, toll?like receptors (TLRs) trigger mitogen activated protein kinase (MAPK) signaling pathways and nuclear factor kappa?B (NF?kB) activation, which regulate NLRP3 expression 11-13 and induce formation of the inflammasome. Its activation then cleaves pro?caspase?1 into the bioactive form, which then induces production of active IL?1β and IL?18; ultimately, this induces pyroptosis, a type of inflammatory cell death 5-9, 14, 15. Therefore, targeting components in the inflammasome pathways may offer a new therapeutic strategy for the treatment of ischemic stroke.
Recently low?level light therapy has gained attraction in treating neurological and psychological disorders because it is relatively cheap, non?invasive, and safe 16-20. Low?level light therapy has been used neurotherapeutically because it can penetrate the scalp and skull 21. In addition, low?level light therapy can modulate a wide range of cellular processes via absorption of light energy via chromophores or photoreceptors in the mitochondria 22. The photochemistry hypothesis is a widely accepted to explain the induction of photobiological effects such as increasing energy in the form of ATP, generating reactive oxygen species (ROS) and nitric oxide, and modification of intracellular organelle membrane activity; these then lead to activation of downstream signaling pathways and transcription factors 23, 24. Transcranial near?infrared light therapy was shown to reduce ischemic brain damage in rabbit acute ischemic stroke 25. Light therapy (710 nm) showed neuroprotection in rat experimental stroke models 26, 27, and has shown clinical promise when tissue regeneration and prevention of tissue damage are required 23. Furthermore, low?level laser light (800 nm) improves cognitive deficits and modulates neuroinflammation after traumatic brain injury 28, 29, and low?level laser therapy (632.8 nm) suppresses microglia activation in BV2 microglial cells 29.
While the use of low?level light therapy mostly involves red and near?infrared light, low?power light emitting diode (LED) using visible light is attractive because LEDs are safer, generating negligible heat at the targeted tissue surface. In addition, LEDs are more affordable, compact/portable, and easier to use. Therefore, we investigated whether acute LED therapy using visible light (orange; 610 nm; see Figure 1 for details on the apparatus) could suppress ischemic brain damage in a focal cerebral ischemia mouse model, using clinically relevant post?stroke parameters.
Experimental scheme of the low?level light emitting diode (LED) therapy. (A) The technical characteristics of the skin?adherent low?level light emitting diode probe. (B) The mice underwent LED therapy (20 min) twice a day for 3 days commencing at 4 h post?ischemia. The control group was kept under isoflurane anesthesia for 20 min without LED application.
All animal experiments were conducted in accordance with the guidelines of the Pusan National University?Institutional Animal Care and Use Committee (PNU?IACUC) on their ethical procedures and scientific care, and were approved by the PNU?IACUC in Pusan National University (Approval Number PNU?2014?0646). Male mice (C57BL/6J, 20–25 g) were housed under diurnal lighting conditions and allowed food and tap water ad libitum. Anesthesia was achieved by face mask?delivered isoflurane (2% induction and 1.5% maintenance, in 80% N2O and 20% O2). Rectal temperature was maintained at 36.5–37.5 °C using a Panlab thermostatically controlled heating mat (Harvard Apparatus, Holliston, MA).
A skin?adherent LED probe (Color Seven Co., Seoul, Korea) was used for LED therapy with the following technical characteristics: peak wavelength, 610 nm (orange color); power intensity, 1.7 mW/cm2; energy density, 2.0 J/cm2 (Figure 1A). Light stimulation was applied by placing the probes (spot size, 4?mm diameter) onto the skin via double?sided tape at two locations on the head (the right midpoint of the parietal bone and the posterior midline of the seventh cervical vertebra) concurrently (Figure 1B). The mice underwent LED therapy (20 min) twice a day for 3 days, commencing at 4 h after the ischemic insult. The control group was kept under isoflurane anesthesia for 20 min without LED (Figure 1B). Experimental drugs including a TLR2 agonist (Pam2CSK4; 50 µg/kg, Invivogen, San Diego, CA) 30, NLRP3 agonist (MSU crystals; 10 mg/kg, Invivogen) 31, and NLRP3 antagonist (MCC950; 10 mg/kg, Sigma, St. Louis, MO) 32 were intraperitoneally administered to mice 30 min before LED therapy. Control mice were administered PBS.
Focal cerebral ischemia was induced by photothrombosis of the cortical microvessels 33. The advantages of this model are simple animal preparation, no craniotomy or mechanical manipulation of cerebral blood vessels or parenchyma, and easily reproducible lesion size and location. Briefly, photochemical dye Rose Bengal (Sigma?Aldrich, St. Louis, MO; 0.1 ml of a 10 mg/ml solution in sterile saline) was injected intraperitoneally so that it entered the blood stream 5 min before illumination. When brain is illuminated by a Cold?light source CL 6000 LED (Carl Zeiss, Jena, Germany), the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption 34. The mice were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) for illumination, the skull was exposed, and bregma and lambda identified. A fiber optic bundle of a cold light source with a 4 mm aperture was centered 2.4 mm laterally from the bregma using a micromanipulator located over the sensorimotor cortex. The brain was illuminated for 15 min, the surgical wound was sutured, and the mice were allowed to recover from anesthesia (Figure 1B).
Mice were deeply anesthetized with sodium thiopental 72 h after ischemic insults, and the brains were removed. The cerebral infarct size was determined on 2,3,5?triphenyltetrazolium chloride (TTC)?stained, 2?mm?thick brain sections. Infarction areas were quantified using the iSolution full image analysis software (Image & Microscope Technology, Vancouver, Canada). To account for and eliminate the effects of swelling/edema, the infarction volume was calculated using an indirect measurement in which the volumes of each section were summed according to the following formula: contralateral hemisphere (mm3) – undamaged ipsilateral hemisphere (mm3).
Neurological deficit was scored in each mouse at 72 h after ischemic insult in a blinded fashion according to the following graded scoring system: 0 = no deficit; 1 = forelimb weakness and torso turning to the ipsilateral side when held by the tail; 2 = circling to the affected side; 3 = unable to bear weight on the affected side; and 4 = no spontaneous locomotor activity or barrel rolling 35.
Mice were deeply anesthetized with sodium thiopental 72 h after the induction of ischemia, and then perfused transcardially with cold PBS. Brain cortices were subsequently collected and total protein was isolated according to the standard methods. Samples were separated by 12% sodium dodecyl sulfate?polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Piscataway, NJ). Immunoblot analysis was performed with the specific primary antibodies followed by secondary antibody conjugated with horseradish peroxidase: TLR?2 (1 : 1000; sc?16237), TLR?4 (1 : 1000; sc?293072), NF?κB p65 (1 : 1000; sc?109), ASC (1 : 1000; sc?22514?R), precursor IL?1β (1 : 500; sc?7884), mature IL?1β (1 : 500, sc?7884), precursor IL?18 (1 : 500; sc?7954), mature IL?18 (1 : 500; sc?7954, Santa Cruz Biotechnology, Dallas, TX), p38 (1 : 1000; 9212S), p?p38 (1 : 1000; 9212S), JNK (1 : 1000; 9251S), p?JNK (1 : 1000; 9251S), ERK (1 : 1000; 4695), p?ERK (1 : 1000; 4695, Cell signaling, Danvers, MA), NLRP1 (1 : 1000; NBP1?54899), NLRP3 (1 : 1000; NBP1?77080), XIAP (1 : 1000; NB100?56183), cleaved caspase?1 (1 : 500; NBP1?45433), pro?caspase?1 (1 : 500; NBP1?45433), cleaved caspase?11 (1 : 500; NBP1?45453), pro?caspase?11 (1 : 500; NBP1?45453, Novus Biologicals, Littleton, CO), myeloperoxidase (MPO, 1 : 1000; af3667, R&D systems, Minneapolis, MN). The intensity of chemiluminescence was measured using an ImageQuant LAS 4000 apparatus (GE Healthcare Life Sciences, Uppsala, Sweden). The membrane was then stripped and incubated with anti?β?actin (1 : 2000; A5316, Sigma) or anti?Lamin B (1 : 1000; sc?3740, Santa Cruz Biotechnology) antibodies as an internal control.
Neuronal death was evaluated by TUNEL analysis and propidium iodide (PI) staining. Mice were perfused transcardially with cold PBS prior to processing of tissue. The frozen brains were cut to a thickness of 8 μm using a CM 3050 cryostat (Leica Microsystems, Wetzlar, Germany), and the TUNEL assay was performed using a DeadEndTM Fluorometric TUNEL System kit (Promega Corporation, Madison, WI). For PI staining, brain sections were incubated with PI (50 μg/ml). After mounting using a fluorescent mounting medium (Vector Laboratories, Inc., Burlingame, CA), images were obtained with a fluorescence microscope (Axio Imager M1, Carl Zeiss). TUNEL(+)/PI(+) cells were counted blindly from three fields per three predefined areas per three adjacent brain sections from each mouse.
Seventy two hours after focal cerebral ischemia, mice were deeply anesthetized with sodium thiopental and subsequently perfused transcardially with cold PBS followed by 4% paraformaldehyde for fixation. Each mouse brain was removed and further fixed in 4% paraformaldehyde at 4 °C for 24 h, followed by cryoprotection in 30% sucrose for 72 h at 4 °C. Next, the isolated brains were frozen in an optimal cutting temperature medium for frozen tissue specimens (Sakura Finetek, Torrance, CA) and stored at –80 °C until examined. The frozen brains were cut to a thickness of 14 μm using a CM 3050 cryostat (Leica Microsystems), and the sections were pretreated with 0.1% H2O2 for 20 min, incubated with blocking buffer (CAS block; Invitrogen Corporation, Carlsbad, CA), and subsequently incubated with primary antibodies against Iba?1 (1 : 200; 019?19741, Wako, Pure Chemical Industries, Osaka, Japan) at 4 °C overnight. The sections were then incubated with biotinylated secondary antibody (1 : 500; BA?1000, Vector Laboratories, Inc.) for 2 h. After several washing, sections were incubated in an avidin?biotinylated peroxidase complex (ABC) reagent (Vectastain ABC kit, Vector Laboratories Inc.) and visualized using a diaminobenzidine (DAB) solution (Vector Laboratories Inc.). All samples were visualized using a light microscope (Carl Zeiss, Jena, Germany). For immunofluorescence staining, the brain sections were immunostained with primary antibodies against MPO (1 : 300; af3667, R&D systems), Iba?1 (1 : 200; 019?19741, Wako, Pure Chemical Industries) or CD68 (1 : 500; MCA1957GA, AbD Serotec, Oxford, UK) at 4 °C overnight. The samples were incubated with FITC? (1 : 500; FI?1000, FI?5000) or Texas Red?conjugated secondary antibodies (1 : 500; TI?9400, Vector Laboratories, Inc.) for 2 h in the dark. The images of each section were captured with a fluorescence microscope (Axio Imager M1, Carl Zeiss) and morphological analysis and quantification of positive cells was conducted using the iSolution analysis software (Image & Microscope Technology). For quantification of positive cells, at least three randomly selected fields (0.36 mm2/field) in the peri?infarct area were examined and averaged. The MPO(+) or Iba?1(+)/CD68(+) cells from three fields per three adjacent brain sections from each mouse were counted.
Quantification of band intensity was performed by Image J software (NIH, Bethesda, MD, USA) and normalized to the intensity of internal control. Data are expressed as the means ± the SEM. Differences between two groups were determined using the unpaired t?test; and comparing more than two groups was determined by one?way analysis of variance (ANOVA) followed by Student?Newman?Keuls test. A P < 0.05 was considered statistically significant.
We first evaluated whether post?ischemic treatment with LED therapy could improve tissue and functional outcomes following focal cerebral ischemia (Figures 1 and 2). As shown in Figure 2A, TTC staining revealed that LED therapy significantly reduced infarct volume relative to controls when measured 72 h after ischemic brain injury (37.0 ± 5.0 mm3 vs. 58.0 ± 7.0 mm3, LED therapy and control, respectively, P < 0.05; Figure 2A and B). Thus, acute LED therapy treatment reduces the spread of ischemic damage. As with infarct volume, ischemia?induced neurological deficits were significantly attenuated in the LED?treated mice (P < 0.05; Figure 2C and Supplementary file), as measured by a scored evaluation of neurologic function (a lower score represents less deficit; see Supplementary file). Together, these findings indicated that acute post?ischemic LED therapy improved tissue?level markers of ischemic damage, and neurological function, in a focal cerebral ischemic mouse model.
Post?ischemic LED therapy improved tissue and functional outcome in a mouse model of ischemic stroke. (A, B) LED therapy (LED?T) reduced infarct volume compared with the control mice (Con). At 72 h after photothrombotic cortical ischemia, brains were removed and brain sections were sequentially obtained. Coronal brain sections (2?mm?thick) were stained with 2,3,5?triphenyltetrazolium chloride (TTC). White indicates the infarct area (A). Quantification of the infarct volume (B) was analyzed using the iSolution full image analysis software (N = 9, * P < 0.05 vs. control group). (C) LED therapy improved neurologic function after cerebral ischemia. Neurological deficit was evaluated 72 h after cerebral ischemia in a blinded fashion followed by neurological score (0 means no deficit. The lower score represents less deficit). Data are expressed as the means ± SEM (N = 9). * P < 0.05 when compared with the control group (Con).
We investigated whether LED therapy modulated ischemia?related neuroinflammation by immunofluorescence staining and western blotting for myeloperoxidase (MPO; Figure 3A–D), a marker of neutrophil infiltration. MPO demonstrated fewer neutrophils in the cortical region after LED treatment (P < 0.01; Figure 3B and C). Western blotting revealed that LED therapy significantly reduced MPO protein levels compared to the control group (P < 0.01; Figure 3D). We next examined microglial activation in the ischemic cortex using Iba?1 (marker protein expressed in both quiescent and active microglia) 36, 37 and CD68 (active microglia marker) 37 using immunohistochemial staining (Figure 3E–G). Iba?1/CD68 double positive cells indicated the active microglia. Iba?1(+)/CD68(+) cells in the ischemic cortex were significantly decreased in the LED therapy group relative to the control group (P < 0.05; Figure 3E and F). Morphology of Iba?1(+) microglia could be more clearly observed in Figure 3G. LED therapy remarkably reduced the Iba?1 immunoreactivies in the penumbra region of the cerebral cortex (Figure 3G). These findings suggest that neuroinflammation such as neutrophil infiltration and microglia activation after ischemic brain injury was effectively rescued by LED therapy.
We further evaluated the effects of LED treatment on neural cell death after focal cerebral ischemia (Figures 4A and 4B). Fewer TUNEL(+)/PI(+) cells (apoptotic cells) were observed in the ischemic cortex of the LED therapy group (P < 0.05; Figure 4A and B). We next examined the effects of LED therapy on levels of inflammasome components in brain tissue ipsilateral to the lesion 72 h after ischemic insult. (Figure 4C). NLRP3 was significantly decreased in the LED therapy group relative to the control group (P < 0.05; Figure 4C). Moreover, LED therapy significantly reduced the levels of cleaved caspase?1 and ?11 (Figure 4D), as well as mature IL?1β and IL?18 in ischemic brain tissue (Figure 4E).
We next investigated whether NLRP3 mediated the in vivo reduction of infarct volume described above (Figure 5). As seen in Figure 5, monotherapy with MCC950, a potent inhibitor of NLRP3 32 reduced infarct volume to sizes similar to LED therapy (Figure 5A and B), although the effect was not statistically significant. In contrast, an NLRP3 agonist (MSU crystals) 31 combined with LED therapy significantly inhibited the reductive effect of LED therapy effect on infarct volume (P < 0.01) (Figure 5A–C). These results indicate that post?ischemic LED therapy decreased ischemic brain damage, possibly by NLRP3?mediated inflammasome suppression.
Activation of TLRs primes NLRP3?mediated inflammasome activation, and thus cell death 38, 39, therefore, we determined expression levels of TLR?2 and TLR?4 (Figure 6A). TLR2 and TLR4 stimulation lead to priming of NLRP3 40, 41. LED therapy significantly reduced TLR?2, but not TLR?4, protein levels in the ischemic cortex (Figure 6A). We also examined MAPKs and NF?kB (Figure 6B and C) protein, as these are components of the TLR pathways. LED therapy significantly attenuated the levels of p?JNK and p?ERK, and significantly reduced translocation of the NF?κB p65 protein subunit into the nucleus, relative to the control group (P < 0.05; Figure 6B and C). These data suggest that LED therapy is capable of decreasing TLR?2?mediated signaling induced by ischemic insult.
Finally, we analyzed whether in vivo infarct volume reduction by LED therapy was mediated by TLR2 (Figure 7). LED therapy significantly reduced infarct volume compared to controls, but when co?treated with the TLR2 agonist Pam2CSK4 30 and LED therapy, the reduction in infarct volume was significantly inhibited (P < 0.001) (Figure 7B). These findings suggested that TLR2 mediated post?ischemic improvements by LED therapy.
These studies determined that post?ischemic LED therapy reduced infarct volume in a focal cerebral ischemia mouse model. We found that LED therapy suppressed neuroinflammation and neural cell death in the ischemic cortex via TLR2?mediated activation and the NLRP3 inflammasome; and that this activation was in turn mediated through MAPK and NF?kB pathways (Figure 8). Notably, we also found improvement in neurological scores after LED therapy.
Interest in low?level light therapy is rapidly growing as new data on its effects are reported 21. Previous reports have demonstrated benefits including rescue of cognitive impairment and other deficits associated with chronic neurological conditions 16-20. Low?level light therapy (633 nm and 870 nm together) has improved cognition in patients with traumatic brain injury 17. Low?level light therapy also improved memory in normal adult rats 19 and middle?aged mice 42. It has been reported that near?infrared light therapy decreases depression in human subjects 16 and improves locomotor activity in rats with traumatic brain injury 18 and mice with Parkinson's disease 20. Moreover, low?level light therapy using near?infrared has reduced ischemic brain damage in experimentally induced stroke in rabbits 25, and showed neuroprotection effect in experimental stroke of rats 26, 27. It was previously reported that low?level light therapy is also effective in a pre?conditioning mode on pain, heart attack, wound healing, central nervous system and so on 43. We recently reported the preventive effect of LED therapy on ischemic brain injury of mouse 44. Since low?level light therapy is economical and has few side effects, it is applicable for clinical prevention, and not just the treatment of the cerebral ischemic disease. While the low?level light therapy mostly focused on red and near?infrared, we are interested in using low?power LED with visible light because LED using visible light are more affordable, compact/portable, and easier to use. Our results were obtained using LED therapy (610 nm orange light) applied twice a day for 3 days, commencing at 4 h after the ischemic event (Figure 2, Supplementary File), and observed the underlying mechanisms of ischemic damage reduction.
For application of light therapy, longer red/near?infrared wavelengths are much better at penetrating tissue than shorter blue/green wavelengths, therefore red and near?infrared lights are preferred clinically. There are few studies to evaluate the transmission rate of radiation in the skull 45, 46. Radiation (emitted in the 600–800 nm spectrum) can penetrate about 1 cm into the skull of human cadavers 45. Jagdeo et al. observed that 600–800 nm radiation range can penetrate soft tissues, bone, and brain parenchyma in cadavers preserved in formalin 46. Although we can suggest the penetration possibility of 610 nm light into human skull from these reports, but we don't know exactly whether our LED parameters are experimentally arrived at the target sites in human. Further investigation needed to clarify this issue.
Ischemic stroke initiates a complex cascade of pathogenetic events that lead to focal brain damage, and inflammation is a major contributor 2. Abulafia et al. 5 described a novel inflammatory mechanism through which the inflammasome contributes to neuronal cell death in cerebral ischemia 5. NLRP3 is known for its role in inflammasome formation, creating multi?protein complexes with ASC and XIAP that are critical for caspase?1 and ?11 activation, and subsequent active IL?1β/IL?18 production 10. During cerebral ischemic injury, there is increased expression of inflammasome components such as NLRP1, NLRP3, ASC, and pro?caspase?1 and ?11 5. While most innate signaling receptors have a relatively restricted ligand spectrum, NLRP3 can be activated by diverse entities such as infectious microorganisms, microbial products, dying cell fragments, and small molecule immune activators 47, 48. It has been suggested that the major role of NLRP3 inflammasomes is in
Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland; Centre de Recherche du Centre Hospitalier de Quebec, Axe Neuroscience et Departement de Medecine Moleculaire de l'Universite Laval, Quebec, G1V4G2, Canada. Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland; Medos International Sarl, a Johnson&Johnson company, Chemin Blanc 38, CH-2400, Le Locle, Switzerland. Medos International Sarl. Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, P.O. Box 5825, Doha, Qatar.
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 alpha-synuclein (alpha-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 alpha-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.
PLoS One 2015 10(10) e0140880
Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland; Centre de Recherche du Centre Hospitalier de Quebec, Axe Neuroscience et Departement de Medecine Moleculaire de l'Universite Laval, Quebec, G1V4G2, Canada. Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland; Medos International Sarl, a Johnson&Johnson company, Chemin Blanc 38, CH-2400, Le Locle, Switzerland. Medos International Sarl. Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar Foundation, P.O. Box 5825, Doha, Qatar.
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 alpha-synuclein (alpha-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 alpha-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.
PLoS One 2015 10(10) e0140880
Federal Institute of Technology (EPFL), Institute of Chemical Sciences and Engineering (ISIC), 1015 Lausanne, Switzerland.
Photobiomodulation (PBM) appears promising to treat the hallmarks of Parkinson's Disease (PD) in cellular or animal models. We measured light propagation in different areas of PD-relevant deep brain tissue during transcranial, transsphenoidal illumination (at 671 and 808 nm) of a cadaver head and modeled optical parameters of human brain tissue using Monte-Carlo simulations. Gray matter, white matter, cerebrospinal fluid, ventricles, thalamus, pons, cerebellum and skull bone were processed into a mesh of the skull (158 x 201 x 211 voxels; voxel side length: 1 mm). Optical parameters were optimized from simulated and measured fluence rate distributions. The estimated mueff for the different tissues was in all cases larger at 671 than at 808 nm, making latter a better choice for light delivery in the deep brain. Absolute values were comparable to those found in the literature or slightly smaller. The effective attenuation in the ventricles was considerably larger than literature values. Optimization yields a new set of optical parameters better reproducing the experimental data. A combination of PBM via the sphenoid sinus and oral cavity could be beneficial. A 20-fold higher efficiency of light delivery to the deep brain was achieved with ventricular instead of transcranial illumination. Our study demonstrates that it is possible to illuminate deep brain tissues transcranially, transsphenoidally and via different application routes. This opens therapeutic options for sufferers of PD or other cerebral diseases necessitating light therapy.
Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand ; Integrative Complementary Alternative Medicine Research and Development Center, Khon Kaen University, Khon Kaen 40002, Thailand. Integrative Complementary Alternative Medicine Research and Development Center, Khon Kaen University, Khon Kaen 40002, Thailand ; Department of Physiology, Neuroscience Program, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
To date, the therapeutic strategy against cognitive impairment in Parkinson's disease (PD) is still not in satisfaction level and requires novel effective intervention. Based the oxidative stress reduction and cognitive enhancement induced by laser acupuncture at HT7, the beneficial effect of laser acupuncture at HT7 against cognitive impairment in PD has been focused. In this study, we aimed to determine the effect of laser acupuncture at HT7 on memory impairment, oxidative stress status, and the functions of both cholinergic and dopaminergic systems in hippocampus of animal model of PD. Male Wistar rats, weighing 180-220 g, were induced unilateral lesion at right substantianigra by 6-OHDA and were treated with laser acupuncture continuously at a period of 14 days. The results showed that laser acupuncture at HT7 enhanced memory and neuron density in CA3 and dentate gyrus. The decreased AChE, MAO-B, and MDA together with increased GSH-Px in hippocampus of a 6-OHDA lesion rats were also observed. In conclusion, laser acupuncture at HT7 can improve neuron degeneration and memory impairment in animal model of PD partly via the decreased oxidative stress and the improved cholinergic and dopaminergic functions. More researches concerning effect of treatment duration are still required.
Bosch Institute, University of Sydney NSW 2006, Australia. Daniel.Johnstone@sydney.edu.au
INTRODUCTION: Previous work has demonstrated the efficacy of irradiating tissue with red to infrared light in mitigating cerebral pathology and degeneration in animal models of stroke, traumatic brain injury, parkinsonism and Alzheimer's disease (AD). Using mouse models, we explored the neuroprotective effect of near infrared light (NIr) treatment, delivered at an age when substantial pathology is already present in the cerebral cortex.
METHODS: We studied two mouse models with AD- related pathologies: the K369I tau transgenic model (K3), engineered to develop neurofibrillary tangles, and the APPswe/PSEN1dE9 transgenic model (APP/PS1), engineered to develop amyloid plaques. Mice were treated with NIr 20 times over a four-week period and histochemistry was used to quantify AD- related pathological hallmarks and other markers of cell damage in the neocortex and hippocampus.
RESULTS: In the K3 mice, NIr treatment was associated with a reduction in hyperphosphorylated tau, neurofibrillary tangles and oxidative stress markers (4-hydroxynonenal and 8-hydroxy-2'- deoxyguanosine) to near wildtype levels in the neocortex and hippocampus, and with a restoration of expression of the mitochondrial marker cytochrome c oxidase in surviving neurons. In the APP/PS1 mice, NIr treatment was associated with a reduction in the size and number of amyloid-beta plaques in the neocortex and hippocampus.
CONCLUSIONS: Our results, in two transgenic mouse models, suggest that NIr may have potential as an effective, minimally-invasive intervention for mitigating, and even reversing, progressive cerebral degenerations.
Object Previous experimental studies have documented the neuroprotection of damaged or diseased cells after applying, from outside the brain, near-infrared light (NIr) to the brain by using external light- emitting diodes (LEDs) or laser devices. In the present study, the authors describe an effective and reliable surgical method of applying to the brain, from inside the brain, NIr to the brain. They developed a novel internal surgical device that delivers the NIr to brain regions very close to target damaged or diseased cells. They suggest that this device will be useful in applying NIr within the large human brain, particularly if the target cells have a very deep location. Methods An optical fiber linked to an LED or laser device was surgically implanted into the lateral ventricle of BALB/c mice or Sprague-Dawley rats.
The authors explored the feasibility of the internal device, measured the NIr signal through living tissue, looked for evidence of toxicity at doses higher than those required for neuroprotection, and confirmed the neuroprotective effect of NIr on dopaminergic cells in the substantia nigra pars compacta (SNc) in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson disease in mice. Results The device was stable in freely moving animals, and the NIr filled the cranial cavity. Measurements showed that the NIr intensity declined as distance from the source increased across the brain (65% per mm) but was detectable up to 10 mm away. At neuroprotective (0.16 mW) and much higher (67 mW) intensities, the NIr caused no observable behavioral deficits, nor was there evidence of tissue necrosis at the fiber tip, where radiation was most intense. Finally, the intracranially delivered NIr protected SNc cells against MPTP insult; there were consistently more dopaminergic cells in MPTP-treated mice irradiated with NIr than in those that were not irradiated. Conclusions In summary, the authors showed that NIr can be applied intracranially, does not have toxic side effects, and is neuroprotective.
We have shown previously that photobiomodulation or near-infrared light (NIr) treatment protects dopaminergic cells of the substantia nigra pars compacta (SNc) in an acute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease (PD). In this study, we tested the protective and rescue action of NIr treatment in a chronic MPTP model, developed to resemble more closely the slow progressive degeneration in PD patients. We examined three regions of dopaminergic cells, the SNc, periaqueductal grey matter (PaG) and zona incerta-hypothalamus (ZI-Hyp). BALB/c mice had MPTP or saline injections over five weeks, followed by a three-week survival. NIr treatment was applied either at the same time as (simultaneous series) or after (post-treatment series) the MPTP insult. There were four groups within each series; Saline, Saline-NIr, MPTP and MPTP-NIr. Brains were processed for tyrosine hydroxylase (TH) immunochemistry and cell number was analysed using the optical fractionator method. In the SNc, there was a significant reduction ( approximately 45%) in TH(+) cell number in the MPTP groups compared to the saline controls of both series. In the MPTP-NIr groups of both series, TH(+) cell number was significantly higher ( approximately 25%) than in the MPTP groups, but lower than in the saline controls ( approximately 20%). By contrast in the PaG and ZI-Hyp, there were no significant differences in TH(+) cell number between the MPTP an MPTP-NIr groups of either series. In summary, exposure to NIr either at the same time or well after chronic MPTP insult saved many SNc dopaminergic cells from degeneration.
Department of Neurology, Medical College of Wisconsin, 8701 W. Watertown Plank Rd, Milwaukee, WI, 53226, USA.
Parkinson's disease (PD) is a neurodegenerative disorder that affects large numbers of people, particularly those of a more advanced age. Mitochondrial dysfunction plays a central role in PD, especially in the electron transport chain. This mitochondrial role allows the use of inhibitors of complex I and IV in PD models, and enhancers of complex IV activity, such as NIR light, to be used as possible therapy. PD models fall into two main categories; cell cultures and animal models. In cell cultures, primary neurons, mutant neuroblastoma cells, and cell cybrids have been studied in conjunction with NIR light. Primary neurons show protection or recovery of function and morphology by NIR light after toxic insult.
Neuroblastoma cells, with a gene for mutant alpha-synuclein, show similar results. Cell cybrids, containing mtDNA from PD patients, show restoration of mitochondrial transport and complex I and IV assembly. Animal models include toxin-insulted mice, and alpha-synuclein transgenic mice. Functional recovery of the animals, chemical and histological evidence, and delayed disease progression show the potential of NIR light in treating Parkinson's disease.
University of Virginia, Morris K Udall Parkinson's Research Center of Excellence and Department of Neurology, Charlottesville, Virginia, USA. firstname.lastname@example.org.
ABSTRACT: BACKGROUND: It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson's disease (PD). Mitochondria supply the adenosine triphosphate (ATP) needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid "cytoplasmic hybrid" neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT). The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC) enhances mitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT).
Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser (50 mW/cm2) for 40 seconds. Oxygen utilization and assembly of mtETC complexes were also determined.
RESULTS: The velocity of mitochondrial movement in PD cybrid neuronal cells (0.175 +/- 0.005 SEM) was significantly reduced (p < 0.02) compared to mitochondrial movement in disease free CNT cybrid neuronal cells (0.232 +/- 0.017 SEM). For two hours after LLLT, the average velocity of mitochondrial movement in PD cybrid neurites was significantly (p < 0.003) increased (to 0.224 +/- 0.02 SEM) and restored to levels comparable to CNT. Mitochondrial movement in CNT cybrid neurites was unaltered by LLLT (0.232 +/- 0.017 SEM). Assembly of complexes in the mtETC was reduced and oxygen utilization was altered in PD cybrid neuronal cells. PD cybrid neuronal cell lines with the most dysfunctional mtETC assembly and oxygen utilization profiles were least responsive to LLLT.
CONCLUSION: The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatment to improve neuronal function in patients with PD.
Morris K. Udall Parkinson's Disease Research Center of Excellence, Department of Neurology, University of Virginia, Charlottesville, VA 22908, USA.
Parkinson's disease (PD) is the eponym attached to the most prevalent neurodegenerative movement disorder of adults, derived from observations of an early nineteenth century physician and paleontologist, James Parkinson, and is now recognized to encompass much more than a movement disorder clinically or dopamine neuron death pathologically. Most PD ( approximately 90%) is sporadic (sPD), is associated with mitochondrial deficiencies and has been studied in cell and animal models arising from the use of mitochondrial toxins that unfortunately have not predicted clinical efficacy to slow disease progression in humans. We have extensively studied the cytoplasmic hybrid ("cybrid") model of sPD in which donor mtDNAs are introduced into and expressed in neural tumor cells with identical nuclear genetic and environmental backgrounds. sPD cybrids demonstrate many abnormalities in which increased oxidative stress drives downstream antioxidant response and cell death activating signaling pathways. sPD cybrids regulate mitochondrial ETC genes and gene ontology families like sPD brain. sPD cybrids spontaneously form Lewy bodies and Lewy neurites, linking mtDNA expression to neuropathology, and demonstrate impaired organelle transport in processes and reduced mitochondrial respiration. Our recent studies show that near-infrared laser light therapy normalizes mitochondrial movement and can stimulate respiration in sPD cybrid neurons, and mitochondrial gene therapy can restore respiration and stimulate mitochondrial ETC gene and protein expression. sPD cybrids have provided multiple lines of circumstantial evidence linking mtDNA to sPD pathogenesis and can serve as platforms for therapy development. sPD cybrid models can be improved by the use of non-tumor human stem cell-derived neural precursor cells and by an introduction of postmortem brain mtDNA to test its causality directly.
Welcome to the laser-therapy.us research tool. This tool is a searchable collection of technical publications, books, videos and other resources about the use of lasers for photobiomodulation. This tool includes almost the entire U.S. library of medicine research papers on LLLT, videos from Youtube associated with therapy lasers and the tables of contents from laser therapy books. This allows users to search for a keyword or condition and see resources about using lasers to treat that condition. All the resources include links to the original source so we are not making any statement about the use of lasers for treating non-FDA cleared application, we are simple summarizing what others have said. Where every possible, we have included a link to the orginal publication.
Here are some of our favorite queries:
This tool uses a broad match query so:
The results of the search are sorted based on 3 quality factors on a scale of 1 to 10 with 10 being the best score. Originally all the resources were given a 5-5-5 until they could be individually evaluated. These scores are purely opinion and are only used to simplify the rank of the results from more valuable to least valuable. This should not be considered a critique of any work. This system was created to help researchers (including ourselves) find the most usable resources for any cold laser therapy research. The resources are assigned values based on the following 3 factors:
Over the past few years of working with research, we found that a majority of the published resources are lacking in one of these three ranking factors.
The original goal of this research tool was to tie published resources to the protocols in the laser-therapy.us library. This connection allows users to trace each protocol back to a list of resources so the protocol can be researched and improved.
When many of the first research papers were published, the most power laser available for therapy were less than 100mW and many systems had to be pulsed to keep the laser from burning out too quickly. Today, system are available that will deliver up to 60,000mW of continuous output. Because of these power limitation, many early studies were limited to extremely low dosages by today’s standards. It takes a 50mW system 17 minutes to deliver 50 joules at the surface of the skin. If this was spread over a large area of damage or was treating a deeper problem, the actual dosages were much less than 1J/cm2. Today, we know that these dosages typically produce very little or no results.
About 80% of the resources in this database are in the near infrared wavelength. There is also some interest in the red wavelength (600 to 660nm) . Other wavelengths like blue, purple, and green have very little scientific research behind them and have not gotten much traction in the core therapy market with the exception of some fringe consumer products.
This research tool is free to use but we make no claims about the accuracy of the information. It is an aggregation of existing published resources and it is up to the user to determine if the source of the resources has any value. The information provided through this web site should not be used for diagnosing or treating a health problem or disease. If you have or suspect you may have a health problem, you should consult your local health care provider.