## 3 Results
### The Presence of NTP-Treated Astrocytes Resulted in Increased Neurite Re-Growth  
Planar primary neuron cultures were plated in 24-well plates, subjected to a scratch assay, and treated with NTP directly. Independently, planar astrocytes were cultured in Transwell plates and NTP-treated prior to being added to the 24-well plate of neurons. Thus, the astrocytes and neurons were cultured together to allow for indirect contact through secretion of factors. Untreated astrocytes co-cultured with neurons subjected to a scratch assay provided the baseline capacity of astrocytes to promote healing. We observed widespread neuronal death in cultures where both astrocytes and neurons were treated with 50 mJ plasma. Therefore, these groups were excluded from further analysis. Growth was observed over time, and at three days following 20 mJ treatment of either astrocytes or neurons (while the other cell type was untreated), there was significantly increased neurite outgrowth (\(p<0.01\)) (Figure 4). Interestingly, at seven days post-treatment, when both neurons and astrocytes were treated with 20 mJ NTP and co-cultured together, neurite outgrowth was not enhanced as compared to the control group (Figure 4E, F). At three days post-treatment (6 DIV), the immunocytochemical analysis confirmed the neuronal phenotype of cells in the 24-well plate and was indicative of neurite outgrowth and the health of neurons in the co-culture system treated with 0 mJ-50 mJ of plasma (Figure 4B).  
Figure 3: Astrocyte stimulation with plasma increased neurite outgrowth under co-culture conditions. **(A)** Phase/Contrast images showing neurite infiltration into the gap area at three days post-treatment (6 DIV) when neurons were treated with 0 mJ plasma and astrocytes were treated with 0 mJ plasma and astrocytes were treated with 0 mJ plasma. Scale: 250 μm. **(B)** Confocal reconstruction of neurons labeled for the neuronal marker, b-tubulin III **(green)** and nuclear counterstain, HOECHST **(blue)** at three days post plasma treatment. Qualitative assessment of cultures indicates that neurons and extending neurites remained healthy three days post-treatment when treated with \(\leq\)50 mJ plasma. Scale: 150 μm. **(C)** Higher magnification of cultures to show increased, healthy neurite outgrowth three days following no plasma treatment of neurons and 20 mJ treatment of astrocytes **(top)**, and unhealthy cultures following 20 mJ treatment of both neurons and astrocytes **(bottom)**. Scale: 100 μm. **(D)** Quantification of remaining gap distance at 6 DIV (three days post plasma treatment). When neurons were left untreated but astrocytes were treated with 20 mJ and vice versa, neurite outgrowth into the gap area was significantly increased compared to the untreated control cultures. **(E)** Phase-contrast images showing neurite outgrowth into the gap area seven days post-treatment (10 DIV) in a subset of treatment groups. Scale: 250 μm. **(F)** Quantification of the remaining gap distance shows that by seven days post-treatment, cultures where neurons were treated with 20 mJ plasma and astrocytes were untreated exhibited a significantly decreased gap distance than untreated cultures. **(D,F)** Bars represent SEM; \({}^{*}p<0.01\).