Transcranial direct current stimulation alters cerebrospinal fluid-interstitial fluid exchange in mouse brain

Abstract

Background: Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has gained prominence recently. Clinical studies have explored tDCS as an adjunct to neurologic disease rehabilitation, with evidence suggesting its potential in modulating brain clearance mechanisms. The glymphatic system, a proposed brain waste clearance system, posits that cerebrospinal fluid-interstitial fluid (CSF-ISF) exchange aids in efficient metabolic waste removal. While some studies have linked tDCS to astrocytic inositol trisphosphate (IP₃)/Ca²⁺ signaling, the impact of tDCS on CSF-ISF exchange dynamics remains unclear.

Hypothesis: tDCS influences the dynamics of CSF-ISF exchange through astrocytic IP₃/Ca²⁺ signaling.

Methods: In this study, we administered tDCS (0.1 mA for 10 min) to C57BL/6N mice anesthetized with ketamine-xylazine (KX). The anode was positioned on the cranial bone above the cortex, and the cathode was inserted into the neck. Following tDCS, we directly assessed brain fluid dynamics by injecting biotinylated dextran amine (BDA) as a CSF tracer into the cisterna magna (CM). The brain was then extracted after either 30 or 60 min and fixed. After 24 h, the sectioned brain slices were stained with Alexa 594-conjugated streptavidin (SA) to visualize BDA using immunohistochemistry. We conducted Electroencephalography (EEG) recordings and aquaporin 4 (AQP4)/CD31 immunostaining to investigate the underlying mechanisms of tDCS. Additionally, we monitored the efflux of Evans blue, injected into the cisterna magna, using cervical lymph node imaging. Some experiments were subsequently repeated with inositol trisphosphate receptor type 2 (IP₃R2) knockout (KO) mice.

Results: Post-tDCS, we observed an increased CSF tracer influx, indicating a modulation of CSF-ISF exchange by tDCS. Additionally, tDCS appeared to enhance the brain's metabolic waste efflux. EEG recordings showed an increase in delta wave post-tDCS. But no significant change in AQP4 expression was detected 30 min post-tDCS. Besides, we found no alteration in CSF-ISF exchange and delta wave activity in IP₃R2 KO mice after tDCS.

Conclusion: Our findings suggest that tDCS augments the glymphatic system's influx and efflux. Through astrocytic IP₃/Ca²⁺ signaling, tDCS was found to modify the delta wave, which correlates positively with brain clearance. This study underscores the potential of tDCS in modulating brain metabolic waste clearance.

Keywords: Astrocytes; Cerebrospinal fluid; Glymphatic system; Interstitial fluid; Transcranial DC stimulation.