Applying the non-invasive light stimulation (LS) therapy directly to the head has shown some beneficial attributes to the diverse neurodegenerative ailing conditions [1]. An virtually incurable neurodegenerative disorder, Alzheimer's disease (AD) has come across as one of the most perturbing health issues, which does not have a promising treatment yet [2]. Photobiomodulation (PBM) is a promising low-power LS which is generally used to stimulate, heal, regenerate, and protect the injured tissues [3]. Therefore, PBM has been overwhelmingly explored in the recent time as a possible means of AD therapy [4,5]. Sometime, photobiomodulation is termed as transcranial low level laser therapy (TLLLT) [6] or transcranial PBM (tPBM) [7], especially when it is employed to correct a cerebral deficit [3]. Essentially, low-powered lasers or light emitting diodes (LEDs) in the red or near infrared (NIR) wavelengths are employed in the photobiomodulation approach in a non-invasive manner to induce the modulation of brain cell function and their metabolic pathways. At present, PBM is regarded as the prevalent LS clinical treatment for AD therapy [8,9], in which not only the cells in nervous and neuroimmune system are activated, but the cerebral electrical activity is also directly influenced by the light irradiation [10].

The observed PBM induced intracellular activity can be primarily attributed to the absorption of light by cytochrome c oxidase (CCO) [11,12]. It was observed that the commonly used PBM wavelengths (i.e., around 650 and 800 nm) actually correspond well with the CCO absorption peaks, which resulted in the emergence of some stimulatory effects with noticeable beneficial attributes concerning the protection of neurons against excitotoxicity [13]. In particular, irradiation by ~800 nm light was shown to induce metabolic shift from glycolysis to mitochondrial activity in pro-inflammatory microglia affected by Aβ peptide, causing increase in the amount of anti-inflammatory microglia and preventing neuronal death [14]. This process is accompanied by an activation of phagocytosis for Aβ clearance [15] and reduction of high levels of reactive oxygen species (ROS) [16], preventing the death of neurons [17]. PBM was found to stimulate brain lymphatics leading to the increase in Aβ clearance [18].It has been also reported that irradiation with NIR light around 1070 nm [19,20], 1210 nm [21] and 1470 nm [22] wavelengths could also induce PBM by different cellular mechanisms. However, the exploration of molecular mechanisms that determine the PBM effects on amyloidogenic disorders are still at early stages. There are only a few reports devoted directly to the mechanism of decreasing amyloids in the brain. Zhan et all discovered that LLLT can reduce Aβ production and plaque formation by shifting amyloid precursor protein (APP) processing towards the nonamyloidogenic pathway by α-secretase (mainly a disintegrin and metalloproteinase domain-containing protein 10, ADAM10) followed by the γ-secretase [23]. A pivotal protein sirtuin 1 (SIRT1) has been found to be involved in this process by specifically up-regulating ADAM10 and down-regulating BACE1, which is dependent on the cAMP/PKA pathway in neurons. These events lead to the decrease in improper beta-secretase cleavage during amyloidogenic APP processing and reduction of toxic amyloid-plaque formation.

Meanwhile, it is well-known that the low gamma activity has a close connection with the appearance of AD [15,24,25]; the accumulation and aggregation of Aβ peptide in the brain can be commonly ascribed to the disruption of gamma oscillations in AD model mice [26]. On the contrary, when the neurons are stimulated by gamma visual stimulation (GVS) to yield higher gamma oscillations, the microglia are also activated, increasing the clearance of Aβ peptide [27]. It was also shown that the activation of the neuroimmune system with 40 Hz pulsed light in AD model mice resulted in the neural activity at gamma frequency that enable the recruitment of microglia to remove Aβ plaques [15].

Despite of the recent surge in the research employing PBM and GVS as the LS means of AD treatment, no comprehensive report compared the treatment efficacy of them. Moreover, it has not been clarified whether pulsed or continuous wave light irradiation would provide better treatment efficacy. The lack of proper standardization of the irradiation parameters [28], along with the undetermined LS mechanisms, posed great hurdle on the path of the progression of LS for treatment of AD. Therefore, exploring the effect of light irradiation parameters on the AD treatment is of great importance for the advancement of this field.

In this context, 808 nm and visible LEDs were applied in continuous wave (CW) and 40 Hz pulsed wave (PW) modes to the in vivo double transgenic (APPswe and PSEN1dE9, APP/PS1) AD model mice in different groups to assess the effect of LS with different light parameters in a systematic manner. The accumulation of Aβ aggregates in brain tissue collected from the LS treated and sacrificed mice was evaluated by epifluorescence microscopy with thioflavine-S fluorescent staining of Aβ aggregates and also by the label-free nonlinear optical imaging [two-photon excited fluorescence (TPEF) microscopy]. Moreover, LS effects have been also evaluated in vivo using combined TPEF and Coherent anti-Stokes Raman spectroscopy (CARS) microscopy to image Aβ aggregates and blood vessels, correspondingly. Moreover, the results of the applied Morris water maze (MWM) test revealed that the spatial learning and memory abilities of AD model mice significantly improved after LS treatment, in contrast to the control, untreated mice. The obtained results provide the useful inputs towards achieve more efficient LS therapy of AD.