Adriano Perez, n/a
Medical Student
Miami Project to Cure Paralysis
Miami, Florida, United States
Stanislava Jergova, PhD
Primary Investigator
The Miami Project to Cure Paralysis
Miami, Florida, United States
Jacqueline Sagen, PhD
Head of Research Laboratory
The Miami Project to Cure Paralysis
Miami, Florida, United States
Our investigation will focus on neuronal mitochondrial dysfunction's role in decreasing GABAergic cell signaling through an in-vitro mechanical damage model and excitotoxicity model. Models will be replicated on a mixed culture of GABA cells with microglia, astrocytes, and a pure GABA cell culture.
Design: Cultures will be derived from embryonic E14 Sprague Dawley rat cortical cells, cultured for 2 weeks, alongside a human GABAergic cell line. Excitotoxicity via exposure to 0.5mM concentrations of glutamate and mechanical injury characterized by a cross being scratched in the center of each well using a sterile micropipette tip. Presence of inflammatory mediators in experimental mixed and pure GABAergic cell cultures via ELISA. Presence of primary antibodies via immunofluorescent staining for GABA (1:200), beta Tubulin (1:1000), and GFAP (1:1000) for cell phenotyping, and MitoTracker for analysis of mitochondrial functions at varying times. Media from cultured cell groups was collected in both groups before the injury then 10 minutes, 24 hours, and 72 hours after the exposure to stress conditions.
Results:
Preliminary results showed GFAP enhancement was only observed in the mixed culture, suggesting glial cell involvement. Increases in IL1b and TNF-alpha were observed in “injured” cells of the mixed GABA culture compared to the control sample of the same culture. The excitotoxic model observed higher levels of inflammatory mediators than the mechanical damage model. Samples collected at times post-injury showed an increase in inflammatory mediators. Reduced signal by MitoTracker was observed in both cell cultures; however, the signal was significantly lower in the mixed culture compared to the pure culture.
Conclusions:
Our findings demonstrate the contribution of glial cells to the post-injury mitochondrial damage of GABAergic cells. Differences observed between mixed and pure culture indicate active participation of glial cells in this process but also offer a platform for further clarification of mechanisms involved in GABAergic mitochondrial dysfunction.
