**3. Effects of RL-TCLT on hippocampal structure and function**

The effects of RL-TCLT on the nervous system, neuronal repair, and improving cognition are growing, and have been well-documented in cellular, animal models, and human studies [8]. Since the past decade, the use of RL-TCLT as an advanced and non-invasive therapeutic method in several brain-related conditions has attracted interest from researchers in biomedical science, including those conditions or pathologies that manifest memory loss. Nevertheless, the underlying neural mechanisms are not well understood. The hippocampus is a brain structure of special importance in studying aging and cognitive decline, its main function is learning and memory [33]. It is a dorsoventrally elongated area, composed of the dentate gyrus (DG), the cornu ammonis (CA) fields CA1, CA2, and CA3, and the subiculum cortex [34]. The trisynaptic circuit is the main excitatory hippocampal synaptic pathway, formed by 3 neuronal groups: granule cells in the DG, and pyramidal neurons of the CA1 and CA3 [35]. This circuit receives inputs from the superficial layers of the entorhinal cortex via the perforant path to the DG. The DG projects to the CA3, which in turn projects to the CA1. Thus, CA1 projects to the deep layers of the entorhinal cortex, closing the circuit [33, 35, 36]. The hippocampus mediates recognition and spatial memory, by a highly regulated circuit with a high-energy demand [37]. Besides, is important to highlight that the hippocampus is highly susceptible to factors such as mitochondrial dysfunction, stress, inflammation, or physiological process such as aging, accumulating damage that gradually lead to a loss of hippocampal function [33].

Spatial memory gradually decreases with the age, since the hippocampus is critical for this type of memory, and the impairment of hippocampal neurons unequivocally results in spatial memory diminishing [37]. Studies from our and other groups have shown the reduced capacity of aged mice to learn and remember spatial tasks [4, 38, 39]. This is indicated by increased time to find a hidden platform in the Morris Water Maze (MWM) or a hidden chamber in the Barnes Maze (BM), two classic probes to evaluate hippocampus-dependent spatial memory [4, 22]. A report using the senescence-accelerated prone 8 (SAMP8) mice, a mouse model widely used to study oxidative impairment, and age-related brain damage, showed that RL-TCLT at 630 nm for two consecutive months prevents spatial memory loss in 5 month-old (mo) SAMP8 mice, and more importantly rescued the cognitive deficits in SAMP8 mice of 7 mo [40]. This last was accompanied by reduced ROS levels in the brain and increased activity of antioxidant enzymes such as catalase and formaldehyde dehydrogenase [40]. Similarly, TCLT with NIR laser at 810 nm applied in mice exposed to acute sleep deprivation showed reduced hippocampal oxidative damage, increasing the activity of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPx) [16]. Additionally, several studies with cells, animals and in clinical trial conclude that RL-TCLT may have a potential effect on the brain since that has been observed that RL-TCTL protect nerve cells from a future impairment, reducing permanent neuronal damage and increasing their survival. For example, the treatment of K369I tau (K3) mice, a transgenic

mouse model of tauopathies and Alzheimer's Disease, with NIR (600–1000 nm) 20 times four weeks reveal a reduction in the size and number of amyloid-β plaques in the neocortex and hippocampus [41].

Besides, RL-TCLT may have a potential effect promoting both synaptogenesis and neurogenesis [42]. Both processes are essential to facilitating connectivity, neural regeneration, and generate structural changes that help to maintain existing neurons, and to encourage the growth of new neurons and synapses process [43, 44]. In this context, IR-light at 808 nm (350 mW/cm2 and 294 J) was applied in the scalp of a photothrombotic model of ischemic stroke in rats for seven days during 2-minute daily. The authors observed that IR-light therapy significantly attenuated behavioral deficits and infarct volume in cortical regions induced by photothrombotic stroke. This improvement was accompanied by neurogenesis and synaptogenesis, as is indicated by increased immunoreactivity of the proliferative and differentiation markers BrdU, Ki67, DCX, MAP2, spinophilin, and the synaptic marker synaptophysin [45]. Also, other clinical studies reveal positive effects of transcranial LED therapy on cerebral blood fluid (CBF) in patients in a vegetative state or with major depression and anxiety. LED treatment by 20 or 30 min per session, thrice per week over 6 weeks, or two times daily for over seventy days, with different wavelengths of 610, 627, and 810 nm increase CBF, improving cerebral vascular perfusion and reducing brain disorders [9, 46, 47]. Similarly, the application of TC-LLL therapy at 810-nm in mice model of cortical impact and traumatic brain injury reveal increased proliferating neural cells around the lesion, possibly activating regenerative mechanisms such as inducing neurogenesis in the dentate gyrus of the hippocampus [48]. Besides, they observed that the mice treated improved learning and memory reducing cognitive impairment [48, 49].

Thus, while its positive effects have been demonstrated countless times in animal models, they have yet to be proven in broad-scope clinical testing. However, the research that does exist is very promising, strongly indicating that RL-TCLT could be a viable treatment for a broad range of neurological diseases including stroke, traumatic brain injury, Parkinson's disease, Alzheimer's disease, and depression, in addition to providing cognitive enhancement for healthy subjects of advanced age that manifest cognitive impairment.
