Photobiomodulation Therapy: in vivo and in vitro Models for Applications in Pediatric Oncology

Photobiomodulation (PBM) Therapy is a form of treatment that exploits laser light to elicit bio-stimulation, promoting cellular metabolism, reducing inflammation, driving analgesia and with an antimicrobial action at blue wavelength. PBM is currently used for the management of oral mucositis (OM) a severe side effect of chemo-radiotherapy characterized by painful atrophic / ulcerative lesions of oral mucosa that can affect pediatric and adult oncologic patients. Although the successful clinical employment of PBM, some issues remain regarding its mechanisms of action, moreover, a universal agreement regarding the most efficacious protocols is still lacking.In this PhD project different aspects of PBM were investigated with the aim of describing the properties of laser light, and consecutively amplifying the scientific knowledge in the laser field.Firstly, PBM effect on oxidative stress was inquired.In OM patients, the clinical parameters improved with PBM at 970nm with the amelioration of clinical scores and with the healing of oral ulcerations, moreover, PBM reduced salivary total oxidant status.In an in vitro model of skin keratinocytes treated with 5-fluorouracil (5-FU), mimicking OM condition, PBM at 970, 905, 800 and 660 nm was able to increment the viability of 5-FU treated cells and to reduce reactive oxygen species (ROS) and gene expression of two antioxidant enzymes.Secondly, PBM was tested for its immunoregulatory actions.PBM was not able to impact on salivary beta-defensins proteins production in OM patients.However, in an oral mucosa epithelial cell line, PBM at 970, 800 and 660 nm reduced mRNA expression of DEFB1, DEFB4, DEFB103 genes. Furthermore, when the cells were pre-treated with lipopolysaccharide, PBM at660nm reduced the expression of DEFB103. Moreover, PBM at 970nm increased IL1B expression and decreased NLRP3 one, meanwhile at 660nm increased NLRP1 expression.Thirdly, the analgesic activities of PBM were investigated.PBM at 970nm was able to decrease the painful sensation referred by OM patients.In primary murine sensory neurons from dorsal root ganglia, PBM at 970 and 800 nm reduced ATP and nitric oxide production and increased ROS level; PBM at 800nm increased also superoxide anion and mitochondrial membrane potential, while at 970nm decreased the calcium flow after capsaicin administration.When capsaicin, as a painful stimulus, were subplantarly injected in mice, PBM at 970nm reduced the pain related behavior.Fourthly, the blue PBM was studied for its antibacterial properties.PBM inhibited Pseudomonas aeruginosa growth on agar plate and in broth, inducing wall damaging. Blue light caused a lethal increment of ROS, that was rescued through a pre-treatment with a ROS scavenger. P. aeruginosa mutants for enzymes of the porphyrin biosynthetic pathway, producing less porphyrins, resulted less susceptible to irradiation.Finally, PBM was able to inhibit bacterial replication in a murine in vivo model of skin abrasion infection. Blue PBM did not result cytotoxic for eukaryotic cells, rather it reduced the inflammatory infiltrate in the murine skin.Lastly, blue PBM was examined for its antiviral activities.Blue PBM was tested on an in vitro model of human skin keratinocytes infected with Herpes simplex virus type 1 (HSV-1).The virus was irradiated alone and then the cells were infected: PBM exerted an antiviral effect, decreasing the viral load and increasing the viability of the cells infected with irradiated virus.The plaque forming unit assay corroborated these results, proven the less infectivity capacity of irradiated HSV-1.So, our results highlighted a pleiotropic action of PBM as anti-oxidant, immunoregulatory, analgesic, antibacterial and antiviral agent. These data corroborated the useful medical interventions based on laser light, possibly helping to increment a clinical thoughtful and justified usage of PBM in OM patients for routinely applications, especially in pediatric oncologic subjects.