## 4 Discussion
When only astrocytes in the co-culture system were treated with 50 mJ of plasma, neurite outgrowth occurred at a rate similar to co-cultures that had not been treated--thus, there was no measurable effect of the plasma. However, when only neurons or only astrocytes were treated with a lower intensity of plasma (20 mJ), significant neurite outgrowth and decreased gap distance were observed (Figure 4D,F). Notably, treating both neurons and astrocytes with 20 mJ plasma did not affect neurite outgrowth (Figure 4). These observations suggest an interplay between the stimulation of one population of cells and the presence of another, such as the presence of healthy astrocytes that are secreting growth factors promoting neurite outgrowth. Since increased gap infiltration was observed when both astrocytes andneurons were treated with low plasma intensities independently, it is possible that (1) low treatments of neurons promoted neurite outgrowth, while little to no effect was due to treatment of astrocytes; (2) treatment of astrocytes led to increased secretion of growth factors, yielding increased neurite outgrowth; and (3) stimulation by plasma treatment promoted both neurite outgrowth and the secretion of factors by astrocytes--the combination of which resulted in increased neurite outgrowth. However, overstimulation may not have beneficial effects as observed by a lack of neurite enhancement following the treatment of both astrocyte and neurons in the co-culture. Therefore, highly mediated NTP treatment intensities must be further investigated and optimized to promote neuronal regeneration. Further studies are needed to determine the mechanism responsible for the phenomenon, such as identifying the optimal levels of plasma treatment on neurons and astrocytes and elucidating the mechanisms within each cell type that are responsible for promoting neurite outgrowth. One possible mechanism by which plasma might enhance neural outgrowth is similar to that seen in stress preconditioning mechanism(s). It is well-documented in the hypoxia/ischemia field that exposure to mild \"mini-insults\" results in the development of injury tolerance, making neurons more resilient to future damage [34]. Preconditioning hypoxic insults have even been reported to enhance axonal regeneration in sensory and motor neurons [35]. Preconditioning is thought to involve a variety of factors including transcription factors and protein kinases, as well as redox-sensitive proteins [34]. It is possible that plasma treatment could also be generating mild mini-insults that activate the same tolerance pathways as hypoxic/ischemic preconditioning. Since non-thermal plasma contains reactive oxygen and nitrogen species, low exposure levels could induce brief oxidative stress resulting in protection from stronger stress at a later time. In fact, [PERSON] and colleagues have reported that pre-treating fibroblasts with a plasma-activated media protected them from later oxidative stress [36]. Along these lines, plasma could perhaps be utilized pre-surgery as a way to promote recovery after damaging procedures; however, future studies will be required to assess the efficacy of any putative preconditioning effects of plasma treatment. Based on our co-culture experiments with NTP-treated astrocytes, plasma may also have utility in the mediation of glial scars, which contribute to the inability of axons in the CNS to regenerate following injury or trauma. A glial scar is comprised of reactive astrocytes that secrete various inhibitory growth factors, leading to defects in the axon growth cone, and, thus, the inability to regenerate. Therefore, glial scars act as both a physical barrier through which axons are unable to penetrate, and a chemical barrier that secretes factors inhibiting axon growth in order to sequester the injured area. There are several factors that are upregulated in the injury micro-environment by many of the cells present in the CNS. These factors include TGF\(\beta\)-1 and -2, which have been implicated in macrophage-induced glial scarring [37; 38].