Photobiomodulation for Parkinson's Disease in Animal Models: A Systematic Review.

Photobiomodulation (PBM) might be an effective treatment for Parkinson's disease (PD) in human patients. PBM of the brain uses red or near infrared light delivered from a laser or an LED at relatively low power densities, onto the head (or other body parts) to stimulate the brain and prevent degeneration of neurons. PD is a progressive neurodegenerative disease involving the loss of dopamine-producing neurons in the substantia nigra deep within the brain. PD is a movement disorder that also shows various other symptoms affecting the brain and other organs. Treatment involves dopamine replacement therapy or electrical deep brain stimulation. The present systematic review covers reports describing the use of PBM to treat laboratory animal models of PD, in an attempt to draw conclusions about the best choice of parameters and irradiation techniques. There have already been clinical trials of PBM reported in patients, and more are expected in the coming years. PBM is particularly attractive as it is a non-pharmacological treatment, without any major adverse effects (and very few minor ones).


Introduction
Parkinson's disease (PD) is a multifactorial and multisystem disease, characterized by the loss of the dopamine producing neuronal cells of the substantia nigra pars compacta (SNc) in the brain [1,2]. The lack of dopamine primarily affects the motor function, but there are many other signs and symptoms that affect mood, cognition, digestive system, sense of smell, etc. The motor symptoms include bradykinesia, muscular rigidity, tremor at rest, and postural instability. The dopamine producing neurons die off, and one somewhat controversial theory to explain this is the accumulation of Lewy bodies containing aggregated α-synuclein inside the cells. The causes of PD are not completely understood. Only about 15% of PD patients are likely to have a genetic cause, among which mutations in leucine-rich repeat kinase 2 (LRRK2), GBA1 (glucocerebrosidase), and SNCA (α-synuclein) are the most common [3]. The environmental causes are complex, but recent evidence has implicated mitochondrial dysfunction [4] and changes in the gut microbiome [5]. Over 1 million individuals in the US suffer from PD and the annual financial burden is estimated to be $52 billion [6]. The accepted treatment is replacement of the lost dopamine using Levodopa, which helps the motor symptoms but

Search Strategy
The primary search was conducted from 1990 to November 2019. Bibliographic databases (i.e., MEDLINE through PubMed, SCOPUS, Web of Science, EMBASE and Cochrane Library) were searched electronically for studies on the neuroprotective effects of PBM on animal models of PD, through the keywords "photobiomodulation", "low-level light therapy", "low-level laser therapy", "near-infrared light", "red light", "Parkinson's disease", and "Parkinsonism". Two independent investigators screened the title, abstract and the full text of the articles and judged the searched materials against the inclusion and exclusion criteria. The search was limited to the original studies performed in animals and to publications written in English. Therefore, ex vivo, in vitro or clinical original articles, as well as review articles were not included.

Inclusion and Exclusion Criteria
We included all in vivo studies reporting the effects of PBM, as opposed to vehicles, on the behavioral and molecular outcomes in PD models. Studies conducting PBM via transcranial, intracranial, systemic irradiation (remotely or laser acupuncture irradiations) as well as whole-body irradiation approaches in PD models were included. Studies performed on ex vivo or in vitro (primary cultures or cell line), as well as clinical trials, were excluded. Additionally, studies conducted on intact (healthy) animals were excluded from our review. Moreover, non-English language publications and studies involving NIR spectroscopy and conference papers were excluded.

Data Extraction
The author, publication year, animals and species, number of animals in each experimental group, gender and age, type of PD model, light source/wavelength, output power, irradiance (power density), irradiation time, fluence (energy density) or energy (dose), total fluence or dose, irradiation approach/site, number of treatment sessions, and outcome(s) were extracted. However, the time of outcome evaluation was not extracted from the studies.

Results
The initial systematic search of the mentioned databases identified 354 articles, of which 28 studies met the inclusion criteria ( Figure 1). Twenty-two articles reported experiments in rodents, five articles reported studies in primates (macaque monkey, Macaca fascicularis), and one study was conducted in a Pink1 mutant PD model. Of the twenty-two studies on rodents, sixteen studies assessed the effects of PBM in mice, of which thirteen were on the albino BALB/c strain and three were on the C57BL/6 strain. Additionally, six rodent studies were performed on rats, of which five were on the Sprague-Dawley strain and one was on the albino Wistar strain. It should be noted that in one study, more than one experiment was conducted using three different animal species of BALB/c mice, Wistar rats, and macaque monkeys; and also in one study, two different types of irradiation methods, transcranial or remote-tissue were performed; in these cases, each experiment was regarded as a separate study and was included in the systematic review.
In fifteen studies, laser or LED light was delivered to the head of the animal in a transcranial approach. On the other hand, nine studies used an intracranial irradiation approach via implantation of an optical fiber connected to a light source into the region of interest inside the brain. In addition, four studies performed systemic PBM using remote-tissue irradiation (abscopal effect) or laser acupuncture methods.  In fifteen studies, laser or LED light was delivered to the head of the animal in a transcranial approach. On the other hand, nine studies used an intracranial irradiation approach via implantation of an optical fiber connected to a light source into the region of interest inside the brain. In addition, four studies performed systemic PBM using remote-tissue irradiation (abscopal effect) or laser acupuncture methods. Whole-body PBM was carried out in one study of Pink1 Drosophila mutant PD model. Eighteen studies applied LED-based devices, while eleven studies used lasers as light sources. Twenty six studies performed PBM with red/far-red wavelengths (627 nm [one study], 630 nm [one study], 670 nm [twenty one studies], and 675 nm [two studies]), whereas, four studies used NIR light (808 nm) and only in one study blue light (405 nm) was delivered via an acupuncture point. The operation mode of light sources in all studies was a continuous wave (CW). Other physical treatment parameters, such as output power, irradiance, irradiation time, fluence, total delivered dose, numbers and duration of treatment sessions are summarized in Table 1. For laser source: increased IL-2 levels no effect on the IL-4, IL-6 and IL-10 levels   Mouse: no effect Rat: no effect Monkey: increased TH + cell number and terminal density in the striatum; increased GDNF expression in the striatum

Discussion
The evidence that has been presented in this systematic review does suggest that PBM (and in particular transcranial PBM) is an effective method to treat animal models of PD. The discovery of the toxic effects of MPTP, which is an impurity found in recreational drugs consumed by individuals in San Francisco in 1982, for the first time allowed the creation of laboratory animal models of PD [52]. Besides MPTP, other compounds have been used to produce PD-like models [53], including 6-hydroxydopamine (6-OHDA) paraquat, rotenone, and Maneb (a polymeric Mn complex of ethylene bis (dithiocarbamate). The mechanism of action of these compounds usually involves metabolism into intermediates that can undergo redox cycling and thereby damage the mitochondria, and in particular Complex 1. There have also been genetic models of PD involving mutations to genes such as α-synuclein, Parkin (an ubiquitin E3 ligase), PINK1 (PTEN-induced putative kinase 1), and LRRK2 (leucine-rich repeat kinase 2). Although the animal models of PD do not completely mimic the human disease, they have been useful for studying the pathophysiology of PD, and for testing the effectiveness of novel treatments, including DBS and PBM. It is expected that further animal studies will use PBM in genetically engineered models of PD rather than toxin-induced models, because these are now considered to be more representative of the human disease.
Although most of animal studies have used red light (670 nm, 675 nm or 630 nm), this does not necessarily mean that red wavelengths are better than NIR wavelengths (810 nm). This preponderance might simply reflect the wider use of red LEDs in ophthalmology and wound healing. The power density levels employed were generally between 20-50 mW/cm 2 , but occasionally lower or higher values were employed. Moderate illumination times (minutes) generally provided fluences in the range of 10-60 J/cm 2 on the scalp. The intracranial fibers that were implanted into the brain delivered fairly low powers (up to 14 mW), but when the illumination was continued for several days, the total energy density delivered could be quite large. It should be noted that the regions of the brain where optical fibers are implanted are different from the regions where electrodes are implanted in the DBS procedure. In DBS, electrodes are usually implanted into the globus pallidus internus to improve the motor function [54] or into the subthalamic nucleus [55] or the caudal zona incerta to improve tremor [56]. The optical fibers in PD animal models have been implanted into the mid-brain, with the goal of delivering the light as close as possible to the SNc, to preserve the dopamine producing neurons.
Pulsing is an interesting parameter for brain PBM therapy, as it has been found that pulsing the light at certain frequencies is more effective than CW light [57]. The two most popular frequencies are 10 Hz (the so-called alpha rhythm) and 40 Hz (the so-called gamma rhythm). The idea is that these frequencies can resonate with intrinsic brain rhythms, and therefore, can improve brain function to a greater extent than CW light [57]. The repetition regimens that have been used for treating the animal models of PD range from a few times per day to every few days, for periods that could be as long as 4 weeks. As PD in humans is a chronic degenerative disease, it is expected that PBM therapy would need to be continued for the foreseeable future.
The encouraging results that have been obtained in the animal studies reviewed above have led to the initiation of clinical studies of PBM therapy for PD patients. Hamilton and colleagues described the construction of "light buckets" lined with LEDs (670, 810 and 850 nm) to treat patients with PD [58] ( Figure 2). These devices delivered a power density of 10 mW/cm 2 to the entire head, and in addition an intranasal device with a power of 4 mW/cm 2 was employed. Patients were treated twice a day (1800 J per session) for 30 days. The initial symptoms of tremor, akinesia, gait, difficulty in swallowing and speech, poor facial animation, and reduced fine motor skills, loss of the sense of smell, and impaired social confidence were all improved in~75% of the subjects, while~25% remained the same and none got worse. The improvements were still maintained over an extended period (up to 24 months). Santos et al. conducted a randomized controlled trial in Parkinson's patients using a CW 670 nm LED array (WARP 10) over 10 cm 2 , on 6 sites on both temples at 60 mW/cm 2 , delivering 6 J/cm 2 and a total energy of 2160 J [59]. A total of 18 sessions were given over 9 weeks leading to clinical improvements. Additional clinical trials are in progress that, in addition to applying light to the head also apply light to the abdomen, with the goal of improving the gut microbiome. The results are eagerly awaited.