Abstract Body: Introduction: Electrical stimulation has proven itself to be a powerful and novel tool in stroke recovery. However, it is unknown why this therapy significantly improves behavioral outcomes in rodent models of stroke. In a hypoxic environment, neural cells lack the proper glucose and oxygen to survive leading to many cell death pathways like Ca²⁺ toxicity. Healthy neural cells can uptake excess intracellular Ca²⁺ through the mitochondria as mitochondrial membrane potential increases. Neural cells under hypoxic conditions, however, are unable to uptake as much calcium due to mitochondrial dysfunction. We hypothesize that electrical stimulation therapy increases mitochondrial membrane potential in neural cells under hypoxia leading to increased Ca²⁺ uptake and less Ca²⁺ toxicity in the cell.

Methods: To test our hypothesis, we used two innovative methodologies: (1) neuron conversion from induced pluripotent stem cells (iPSCs) with neurogenin-2 (Ngn2) overexpressed for a period of 28 days developed in the Wernig Lab, and (2) a custom in vitro stimulation setup consisted of a gold layer sputtered onto cell culture inserts and a copper sheet placed below the 24-well plate creating an electric field across the iN cells (Figure 1). Induced neurons (iN) underwent oxygen glucose deprivation (5% CO_2, 10% H₂, and 85% N₂) for a period of four hours in glucose deprived media. One hour after exposure, iN cells were electrically stimulated at 50mV, 100Hz, and a 20% duty cycle for 20 minutes at 37°C. One day after stimulation, iN cells were stained with Rhod-2, a labeled calcium indicator that increases in fluorescence as a function of mitochondrial Ca²⁺, and DAPI to confirm cell presence.

Results and Conclusions: Stimulation significantly increased mitochondrial Ca²⁺ concentration in healthy iN cells seen in Figure 2 (N = 5, p = 0.014). In Figure 3, stimulation also significantly increased mitochondrial Ca²⁺ in hypoxic cells (N = 5, p = 0.0024). Increased mitochondrial Ca²⁺ in both cases indicates that mitochondrial calcium uptake appears to be improving. Electrical stimulation increasing mitochondrial membrane potential and improving calcium uptake could be a mechanism by which electrical stimulation facilitates stroke recovery. Future work will include additional staining for cytosolic calcium concentration (Calcium Green) and mitochondrial membrane potential (TMRM) to better track the relationship between the two.