A study of primary sensory neurons and Schwann cells sensitivity to visible light irradiation in vitro, and feasibility of a light inducible system in Schwann cells for neurite outgrowth enhancement in vitro

Abstract

Peripheral Nerve Injuries (PNIs) are serious and debilitating conditions that lead to the mechanical and sensorial dysfunction of limbs [1]. Despite clinical intervention, prognosis is poor and only 50% of the patients achieve functional recovery [2]. Nevertheless, recent advances in understanding the fundamentals of PNI biology have guided researchers to develop therapies to treat nerve injuries using genetically engineer Schwann cells, the glial cells of the peripheral nervous system (PNS) [3-8]. However, the absence of an activation-deactivation feature in these experimental approaches can lead to undesired side effects such as prolonged high concentrations of growth factors and abnormal accumulation, nerve fiber entrapment, or constrain Schwann cells' re-differentiation which is required for functional recovery after injury [8, 9]. Therefore, this doctoral research focused on study the feasibility of implementing a light inducible approach to reversibly modulate Schwann cell phenotype with the goal of promoting neurite outgrowth. As first step towards this goal, multi-well plate Light Emitting Diode (LED) arrays were established to irradiate primary cell populations at different wavelengths and doses. Characterization of the effects of light irradiation in primary Dorsal Root Ganglion (DRG) neurons and Schwann cells indicated that the sensitivity of primary DRG neurons to visible light irradiation varied with wavelength and doses applied in vitro. Light irradiation at 470 nm and doses equivalent or above 9.0 J/cm2 reduced neurite outgrowth compared to neurons kept in the dark, while neurons exposed to light at 627 and 567 nm remained unchanged. Despite these findings, no signs of neuronal cell death or apoptosis were observed. In contrast, Schwann cells were insensitive to light irradiation between 627 and 470 nm with metabolism, proliferation, and mRNA levels of master regulator transcription factors c-Jun and krox-20 remaining unaltered after irradiation. The results indicate different sensitivities to light irradiation between neurons and glial cell from the PNS. Afterwards, a light inducible transcription tool (LITEZ) was selected and customized to manipulate the expression of the transcription factor element c-Jun, important for the modulation of pro-regenerative and mature phenotypes in primary Schwann cells. Transiently modified Schwann Cells were induced to express exogenous c-Jun via light irradiation at 470 nm wavelength. However, when genetic modification aimed to establish a stable expression of LITEZ elements in Schwann cells, light induction failed due to loss of expression of the proteins needed for the light inducible system even when genome integration of cassettes were achieved and antibiotic resistance was present. Therefore, using LITEZ as a tool for gene modulation in the regeneration of peripheral nerves is not a feasible strategy due to tissue wavelength sensitivity and lack of expression in primary Schwann cells.