Direct Current Stimulation Modulates Gene Expression in Endothelial Cells and Astrocytes

Abstract

Transcranial direct current stimulation (tDCS) is currently investigated for treatment of neurological disorders, as an aid in rehabilitation, and to facilitate cognitive performance in healthy individuals. The cellular mechanisms explaining such a diversity of applications remain under investigation. While previous work has focused on the effect of tDCS on neurons, little is known about the effects of tDCS on other cells in the brain, such as the endothelial cells (EC) in brain blood vessels and the astrocytes that wrap around them. The objective of this study was to investigate the potential effect of tDCS on the gene expression of ECs and astrocytes that form the blood-brain-barrier. For this purpose, Direct Current Stimulation (DCS) was applied to monolayers of immortalized mouse brain ECs (bEnd.3) or human astrocytes in a specially designed chamber that generated spatially uniform current. There were four groups in each experiment: control static (con S), control pressure (con P), DCS pressure (DCS P), and DCS static (DCS S). The pressure groups were exposed to a hydrostatic pressure gradient that induced flow across the monolayers. DCS was achieved by means of a pair of Ag/AgCl electrodes. A transcranial Direct Current Stimulator was used to apply 0.1 or 1 mA across the monolayers for 10 minutes. RNA was collected immediately or 1-hr after DCS and reverse transcribed. The resulting cDNA was use in Real Time-Polymerase Chain Reaction (qPCR) to look at the gene expression of a set of neuroactive genes. Pressure-driven flow alone (con P) induced moderate but significant gene expression changes in 8 of the 13 genes studied in bEnd.3 cells. Of note, BDNF, a molecule that regulates synaptic plasticity and promotes survival of nerve cells, had a 2-fold increase in expression with flow. DCS plus pressure-driven flow (DCS P) did not induce significant changes compared to con P at either current magnitude. DCS alone (DCS S; 1mA) resulted in a 2.4-fold upregulation of FGF9, an important growth factor for glial cells. In addition, DCS S (1mA) induced downregulation (0.55-fold) of NTF3, a growth factor involved in nerve cell differentiation and survival. In astrocytes, 9 out of 21 genes studies had significant changes under con P conditions. DCS alone also induced significant gene expression changes in 9 of the 21 genes. BDNF was downregulated (0.78) by pressure-driven flow and upregulated by DCS at both current magnitudes (1.22 and 1.40 at 0.1 and 1mA, respectively). BDNF upregulation was enhanced when RNA was collected 1-hr after DCS (1.77 and 2.33 at 0.1 and 1mA, respectively). The largest change for astrocytes under DCS S conditions was on FOS, a marker of neuronal activity, which was upregulated by 10-fold and 3.3-fold at 0.1 and 1mA, respectively. This study demonstrates that both flow and DCS can directly modulate gene expression of endothelial and astrocyte cells in the brain. The results suggest new adjunct mechanisms that may contribute to the aggregate changes in neuronal activity and learning produced by tDCS.