## 4 Discussion  
Nervous system trauma and neurodegeneration often result in permanent functional deficits due to the limited regenerative capacity of the brain and spinal cord. In the current study, we assessed the effects of non-thermal plasma treatment on neurodegeneration by performing an in vitro scratch assay on monolayer cultures of astrocytes, neurons, and astrocyte-neuron co-cultures. Immediately following the scratch, cultures were treated with nspDBD plasma, and the effect of treatment on astrocyte re-growth/migration or neurite outgrowth was measured. In the past, typical DBD plasma systems used for biological applications generated non-uniform plasma, characterized by a filamentary structure. These structures are potentially problematic as they may cause localized heating and damage to the surfaces they contact [29; 30]. This was largely a result of the applied electric field in the discharge gap as well as the topographically uneven treatment surfaces, such as tissue [29; 31; 30]. Development of the more sophisticated DBD system, which supplied high voltage over nanosecond rise times was shown to generate a more uniform plasma over uneven surfaces [29; 31]. Our group has previously characterized energy-dependent responses of several different cell types (e.g., epithelial, fibroblast, macrophage, etc.) to NTP used in this study [11; 28; 32; 33]. These studies established the plasma parameters and treatment energies used in the present work for stimulation of migration and neurite outgrowth of neurons and astrocytes with minimal lethality. When astrocyte monolayer cultures were treated with plasma, the infiltration of astrocytes into the gap area was seen over time, as expected (Figure 2B). This suggests that at a lower intensity plasma treatment, cell viability or proliferation were not affected. Interestingly, cultures treated with 50 mJ plasma consistently yielded decreased mean gap distance when compared to time-matched untreated control cultures (Figure 2B). These results suggest that plasma treatment of astrocytes may have persistent effects that lead to increased astrocyte proliferation, migration, or hypertrophy, consequently leading to increased gap closure. Although the mechanism by which this phenomenon is occurring requires further investigation, these results suggest that plasma treatment has a direct effect on astrocytes, which may impact neuron or neurite outgrowth. Due to the positive effects on cell proliferation and migration seen in astrocyte cultures, as well as the known ability of plasma treatment to increase secretion of growth factors in vitro, direct plasma treatment of neurons was also analyzed. It should be noted that neurite outgrowth, and not neuronal proliferation, was the chosen metric due to the negligible proliferative capabilities of primary cortical neurons, though our measurements also account for any migration of the neuronal somata at the periphery of the scratch zone. Neurite outgrowth did not statistically differ between plasma-treated groups (Figure 3D,F), but a trend was present, suggesting 10 mJ and 20 mJ plasma treatment resulted in greater--and denser--neurite outgrowth and gap closure (Figure 3). A similar trend was observed at a later time point post plasma treatment. While it is possible that direct plasma treatment did not affect the rate of neurite outgrowth, further investigation into the effects of plasma treatment and optimal frequencies on neurons and axons, as well as longer-term implications may be required. As discussed, direct stimulation of astrocytes resulted in increased infiltration, and the plasma treatment of neurons did not yield a decrease in neurite outgrowth. Therefore, a co-culture system was utilized in which neurons and/or astrocytes could be plasma treated independently, and where the cells do not come in direct contact with one another. Here, neurons in the lower chamber were only exposed to the factors secreted into the media by astrocytes due to the semi-permeable membrane.