Non-Thermal Plasma Accelerates Astrocyte Regrowth and Neurite Regeneration Following Physical Trauma In Vitro

Abstract

Non-thermal plasma (NTP), defined as a partially ionized gas, is an emerging technology with several biomedical applications, including tissue regeneration. In particular, NTP treatment has been shown to activate endogenous biological processes to promote cell regrowth, differentiation, and proliferation in multiple cell types. However, the effects of this therapy on nervous system regeneration have not yet been established. Accordingly, the current study explored the effects of a nanosecond-pulsed dielectric barrier discharge plasma on neural regeneration. Following mechanical trauma in vitro, plasma was applied either directly to (1) astrocytes alone, (2) neurons alone, or (3) neurons or astrocytes in a non-contact co-culture. Remarkably, we identified NTP treatment intensities that accelerated both neurite regeneration and astrocyte regrowth. In astrocyte cultures alone, an exposure of 20-90 mJ accelerated astrocyte re-growth up to three days post-injury, while neurons required lower treatment intensities (≤20 mJ) to achieve sub-lethal outgrowth. Following injury to neurons in non-contact co-culture with astrocytes, 20 mJ exposure of plasma to only neurons or astrocytes resulted in increased neurite regeneration at three days post-treatment compared to the untreated, but no enhancement was observed when both cell types were treated. At day seven, although regeneration further increased, NTP did not elicit a significant increase from the control. However, plasma exposure at higher intensities was found to be injurious, underscoring the need to optimize exposure levels. These results suggest that growth-promoting physiological responses may be elicited via properly calibrated NTP treatment to neurons and/or astrocytes. This could be exploited to accelerate neurite re-growth and modulate neuron-astrocyte interactions, thereby hastening nervous system regeneration.Each year in the US, more than 80,000 people are afflicted with severe traumatic brain injury (TBI), over 12,000 experience spinal cord injury (SCI), over 500,000 people suffer from stroke, and millions of people are diagnosed with chronic neurodegenerative diseases [2,3]. Following brain injury or neurodegenerative disease, successful regeneration often requires the reformation of long-distance communication fibers, referred to as axons. Long-distance axonal regeneration does not occur in the mature brain, and the formation of a glial scar, occurring after a severe injury, inhibits axon regeneration and cell migration [4]. Strategies to augment axonal regeneration and modulate the post-injury microenvironment are actively being investigated to promote healing and facilitate functional regeneration.Non-thermal plasma (NTP), sometimes referred to as cold atmospheric plasma, has been investigated to facilitate wound healing and promote tissue regeneration. It is a partially ionized gas that consists of several physical (e.g., electric fields, ultraviolet light) and chemical (e.g., radical species and charged and neutral molecules) constituents [5]. NTP has...

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