**4. Effects of RL-TCLT on synaptic neurotransmission and synaptic plasticity**

Considering that irradiation with RL between 600 and 1200 nm produces changes at molecular, cellular, and tissue levels [50, 51] improving cognitive capacities [52], this enhancement in brain function will result in synaptic neurotransmission and synaptic plasticity potentiation after light treatment [53, 54]. Neurotransmission and synaptic plasticity represent the capacity of synaptic connections to adapt structurally and functionally in a stimulus-dependent manner [43]. Both synaptic neurotransmission and synaptic plasticity can be affected by different factors, such as mitochondrial dysfunction and increased oxidative stress, as well as physiological events including aging, stroke, brain injuries, or neurodegenerative disease, among others [22, 55, 56]. Therefore, treatments focused in maintain or promote neurotransmission and synaptic plasticity are attracting increasing attention. In this context, despite the beneficial effects showed for RL-TCLT on cognition, practically not exist electrophysiological studies using this therapy. As an approximation, we will discuss the studies using transcranial lowlevel laser light (TC-LLL).

### *Transcranial Red LED Therapy: A Promising Non-Invasive Treatment to Prevent Age-Related… DOI: http://dx.doi.org/10.5772/intechopen.100620*

Studies both *in vitro* and *in vivo* have shown that TC-LLL therapy supports neural function, this has been observed principally in reports using transgenic mouse models of Alzheimer's disease (AD) [57]. Meng et al. observed that TC-LLL therapy at 632.8 nm in primary hippocampal neurons treated with full-length Aβ1–42 peptide reduced Aβ-induced neurotoxicity. In addition, TC-LLL therapy shows neuroprotective effects decreasing Aβ-induced dendrite atrophy [57]. Also, TC-LLL treatment increased the expression of brain-derived neurotrophic factor (BDNF) in cell line and cultured neurons derived from APP/PS1 transgenic mice, suggesting that this neurotrophin will be modulating dendritic structure, promoting the survival of neurons and dendrite growth, and potentiating synaptic transmission in the CNS [57, 58]. All these results can be explained by the activation of the ERK/CREB/ BDNF pathway mediated by TC-LLL therapy [45] because this pathway is involved in the dendritic development of neurons [45, 57, 59]. Therefore, the TC-LLL therapy can induce activation of signaling transduction pathways, and gene transcription, which increases protein expression of different synaptic effectors and modulators, effects that also are potential therapeutic in treating neurodegenerative disease.

Interestingly, the NIR-LED light treatment at 670nm in the Tg2576 mice model of AD, which progressively accumulated Aβ in their brain [60], indicate that NIR-LED therapy decreased the levels of Aβ1–42 at the synapses and Aβ oligomer-induced reduction in long-term potentiation (LTP), relevant processes of neuroplasticity that correlates with memory formation [61]. Therefore, NIR-LED light therapy recovered crucial processes related to synaptic function, necessary to the preservation of cognition abilities [62]. Additionally, studies with photobiomodulation transcranial therapy with wavelengths of 635nm in a mouse model of depression showed that this treatment reduces glutamate levels and neurotoxicity, improving the depressant behavior. These beneficial effects can be explained by the activation of the PKA pathway and the increased levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In addition, reduce the expression of GluA1, decreasing the glutamatergic neurotransmission. Thus this therapy could rescue excitatory synaptic transmission, improve synaptic plasticity, and have also a potential anti-depressive effect [63].

Thus, several pieces of evidence suggest that the application of transcranial therapy light could be used to improve cellular components associated with the synaptic function [42], which is essential in the maintenance and preservation of cognition, including learning and memory [43]. In addition to the therapeutic effects at the molecular level, it is proposed the generation of changes at the behavioral level, such as cognitive improvement, antidepressant effects, and sleep improvement [42]. Furthermore, this therapy can stimulate neuronal organization or reorganization, therefore it could be extremely promising as a method of stabilization and/ or improvement of various brain disorders or nervous system, and neurodegenerative diseases [30, 42]. However, more extensive studies are necessary to evidence all the cellular and molecular mechanisms involved in these encouraging results. This last especially considering that the evidence summarized here consider severe differences in the device and light type used, the protocol of administration, and the study model. Is imperative to advance understanding the multiple targets of red light in the synaptic structure and function.

## **5. RL-TCLT mechanisms: improving mitochondrial function**

Considering the multiple reports revised previously, now known that Red Light therapy, including Red and InfraRed LED light and Red Laser light, have favorable effects on brain structure and function, and especially in the hippocampus

improving cognitive functions [9, 25, 42]. This enhances in cognitive capacity could be explained by the activation of neurogenesis and synaptogenesis [9, 42], as well as by the stimulation of processes related to synaptic plasticity such as LTP [62]. However, any of these events reveal a potential mechanism by which Red light treatment results therapeutic to different affections such as aging, neurodegenerative disease, stroke, and depression among others [42].

Interestingly, all pathological conditions mentioned previously involve, almost in part, dysfunction of hippocampal neurons attributable to mitochondrial defects [64, 65]. For example, mitochondrial dysfunction is considered a hallmark of aging and could be considered one of the factors leading to neurodegeneration [4, 22, 66]. Studies in humans and animal models showed that decreased memory correlates with reduced cerebral energetics metabolism and more specifically to mitochondrial bioenergetics deficits [4, 22, 66]. Therefore, the mitochondrial focus of aging and neurodegenerative diseases is of great interest for the development of a potent and ideally non-invasive anti-aging intervention to improve or attenuate cognitive impairment in the elderly.

Notably, enhanced metabolic functioning is one of the most identifiable properties of irradiate neuronal cells with RL or NIR light, resulting in increased intracellular ATP production [8]. Thus, mitochondrial ATP production is one of the most strongly suggested mechanisms of action of RL therapy [5, 8]; for example, studies using RL-TCLT at 660 nm for 15 sec daily for 2 weeks in aged 18 mo mice improved ATP concentration [16]. More specifically, studies in vitro with RL and NRL LED radiations with a wavelength between 600 and 850 nm have shown that the effects of this treatment are principally attributed to photon absorption by complex IV of the mitochondrial respiratory chain [5]. This mitochondrial complex corresponds to the cytochrome c oxidase (COX) enzyme [22] and it seems that RL increases the activity of this enzymatic complex, leading to enhancement of oxygen consumption and ultimately to mitochondrial respiration [5]. COX is a photo acceptor of RL and NIR light, which generates a redox change in the enzyme [5, 8]. In turn, this causes a transient change in mitochondrial membrane potential (mψ) and increases ATP production [5, 16]. Thus, wavelengths corresponding or near to red will be improving the mitochondrial production of ATP, potentiating the synaptic and cognitive function [5, 42]. Nevertheless, is important to highlight that other works report that RL could inhibit the COX enzyme. In particular, NIR wavelengths of 750nm and 950nm reduced the activity of the COX complex. This results in decreased mitochondrial respiration and a loss of mitochondrial membrane potential (ΔΨm) [67]. Is surprising to note that the attenuation of mitochondrial function and the concomitant production of superoxide radical reduce neuronal death exposed to oxygen–glucose deprivation and in a mice model of ischemia, an effect that is not observed after other NIR wavelengths that activate COX [67]. Altogether, these contradictory results question the real effect of RL on COX responsible for the beneficial effects of this therapy (**Figure 2**).

On the other hand, several reports showed that RL-LED modulates the levels of reactive oxygen species (ROS) [30, 40]. Studies using RL-LED illumination at 630 nm reduces brain H2O2 levels in cultured cells and the brain of SAMP8 mice [40]. This could be explained by an increment in the activity of antioxidant enzymes such as catalase or also could be a consequence of increased mitochondrial function with reduced electron leak [5, 16]; more studies are necessary to evaluate these possibilities. Besides, in this study, the authors showed that RL-LED absorption activates transcription factors that regulate long-lasting effects on gene expression [16], therefore this suggests that Red Light therapy could be a more complex mechanism, at a long time, and not only a transient activation of several enzymes. Other results also showed that Red 635 nm irradiation inhibits the expression of COX *Transcranial Red LED Therapy: A Promising Non-Invasive Treatment to Prevent Age-Related… DOI: http://dx.doi.org/10.5772/intechopen.100620*

### **Figure 2.**

*Mechanism of action proposed to red and near-infrared light and its cellular effects. Transcranial therapy using red and near-infrared light has been proposed to photoactivate the cytochrome c oxidase (COX) enzyme, the complex IV of the electron transporter chain of the mitochondria. However other reports propose that several wavelengths inhibit COX enzyme; modulating ROS and ATP production, calcium homeostasis, and inflammatory processes.*

enzyme, reducing ROS levels and mRNA of cytosolic phospholipase A2 (cPLA2) and secretary phospholipase A2 (sPLA2) [68]. This also consequently inhibits the release of PGE2, suggesting an additional anti-inflammatory effect (**Figure 2**).

Additional mechanisms that will be involved in the positive effects of RL-LED implicate Ca2+ ions modulation [8, 40]. RL and NIR LED are recognized by water groups formed in the heat/light-gated Ca+2 channel. This induces vibrational water energy, which in turn disorganizes the protein structure of the Ca+2 channel. This conformational change finally leads to channel opening; modulating intracellular Ca+2 levels [5]. This possible mechanism is relevant in neurons, considering that intracellular Ca+2 levels are critical to trigger survival or death pathways related to synaptic activity [69].

In summary, despite various mechanisms that could be mentioned such as the potential molecular target of RL and NRL, still is necessary additional research in the field to understand the events that result in synaptic and cognitive function. Possibly these improvements are the result of diverse events occurring simultaneously.

## **6. Future perspectives of transcranial Red630-light-transcranial LED therapy preventing age-related memory loss: Our advances**

Despite diverse studies shown possible molecular targets of RL-LED therapy [5, 8], the precise mechanism underlying the neuroprotective actions of RL-TCLT is not completely understood. Therefore, more studies are required to determine the

biological events that lead to neuroprotection or neuronal repair in both aging and neurodegenerative diseases. Possibly, the main problem related to the incapacity of determining a detailed mechanism is based on the variability of wavelengths, times of treatment, and models used [15, 16, 40, 63, 70]. Therefore, this highlights the need for complete studies using the same mice model, LED dispositive, and therapy protocol, to understand and describe the mechanism(s) underlying the benefits of RL-TCLT.

Interestingly, RL-TCLT at 630 nm in patients with traumatic brain injuries, using a helmet that emits radiation for 30 min, three times per week, for six weeks showed a great reduction in post-traumatic stress symptoms, insomnia, and depression, suggesting improved cognitive function [71]. More importantly, the same RL-TCLT used in aged patients with mild cognitive impairment improves memory in these aged humans. For this reason, and to study the complete effects and mechanisms of RL-TCLT in aging, we designed a unique RL-TCLT device to emit homogeneous light at a wavelength of 630 nm, with 100 J of energy, a power density of 0,35 w/ cm2 , and an energy density of 43.5 J/cm<sup>2</sup> in the brain of mice, specifically in the hippocampus.

We applied RL-TCLT to the hippocampus of 7.5mo SAMP8 mice, a mice model of accelerated aging, with an irradiation time of 125 s daily (excluding weekends) for 5 weeks. This protocol is equivalent to the applied to patients with mild cognitive impairment described previously, and the mouse lifespan. We started the RL-TCLT in SAMP8 at 7.5mo because we and other authors showed that the non-transgenic SAMP8 mice present age-related hippocampal memory loss since 6mo and is more evident from 7mo onwards [72]. Interestingly, our results reveal that 7.5mo SAMP8 mice treated with RL-TCLT at the hippocampus improves spatial learning and memory of aged SAMP8 mice. This cognitive improvement will be due to a possible remodeling of the synaptic structure toward more active synapses reducing the risk of excitotoxic events. This is suggested by i) an increase in presynaptic proteins such as synaptophysin (SYP) and Synapsin (SYN) that increase the neurotransmitter release [73], ii) a decrease in the NMDAR subunit NR2B, whose protein levels are related to excitotoxicity [74] and iii) higher Arc protein levels, a marker of synaptic plasticity [75] (Jara et al., manuscript in preparation).

Considering that both memory formation and synaptic activity are highly dependent on energy [76], that mitochondria are the main ATP producer of the cell [22], and that the suggested mechanisms by RL-TCLT target the mitochondria [5], we evaluated different mitochondrial functions in the hippocampus of treated SAMP8 mice with RL-TCLT. Relevantly, we observed increased ATP production, higher activity of the OXPHOS complex II-III, and IV (COX enzyme); suggesting that RL-TCLT directly stimulates mitochondrial bioenergetics function enhancing the activity of other OXPHOS complexes in addition to COX (Jara et al., manuscript in preparation). Similarly, we observed decreased levels of the mitochondrial calcium uniporter (MCU), suggesting that it will result in reduced mitochondrial Ca+2 overload and swelling, enhancing mitochondrial Ca+2 buffering. In fact, this last was validated in Ca+2 overload assays in hippocampal mitochondria from RL-TCLT SAMP8 mice (Jara et al., manuscript in preparation), indicating that RL-TCLT also improves the calcium buffering capacity of the aged hippocampal mitochondria. Whether bioenergetics and calcium buffering enhancing are directly related or are independent mechanisms requires future analysis.

Thus, our results indicated RL-LED-mediated mitochondrial stimulation, which could be transient or permanent. But it is difficult to think that only transient activation of mitochondrial function could explain the improved cognitive effects produced by RL-TCLT treatment. Although mitochondrial ATP production is vital for synaptic communication, it is probably not solely sufficient to

*Transcranial Red LED Therapy: A Promising Non-Invasive Treatment to Prevent Age-Related… DOI: http://dx.doi.org/10.5772/intechopen.100620*

result in improved hippocampal memory. Therefore, is highly probable that other mechanisms are involved in the beneficial effects of RL-TCLT, which result in gene transcription and the consequent cellular remodeling. In concordance with the anterior, we also observed higher PGC-1α protein levels, a transcriptional coactivator considered the main inducer of mitochondrial biogenesis that also regulates mitochondrial function [77]. This suggests that RL-TCLT will stimulate the generation of new mitochondria or the activation of gene-dependent mitochondrial reparation pathways that result in increased mitochondrial function. However, this requires a robust study.
