Targeted temporal interference stimulation of the lateral hypothalamus improves glymphatic function and neurological outcomes after traumatic brain injury

Glymphatic system (GS) dysfunction contributes to impaired solute clearance, neuroinflammation, and secondary injury after traumatic brain injury (TBI). We investigated whether temporal interference stimulation (TIS) targeting the lateral hypothalamus (LH) improves neurological outcomes and GS function after TBI and explored the underlying mechanism. Finite element modeling was used to optimize the electrode montage and stimulation parameters for LH-targeted TIS. Treatment effects were assessed by behavioral testing, histopathology, glymphatic tracer imaging, and neurovascular evaluation. LH activation, Orexin A (OXA)/orexin receptor 2 (OX2R)-CaMKII signaling, and aquaporin 4 (AQP4) polarization were examined using immunofluorescence, transcriptomic profiling, molecular analyses, and pharmacological pathway inhibition. Mechanical stretch injury and astrocyte-neuron or astrocyte-endothelial co-culture models were used for further mechanistic validation. LH-targeted TIS increased LH neuronal activation with spatial specificity and no detectable short-term adverse effects, improved neurological and cognitive performance, and reduced neuronal injury, cerebral edema, and neuroinflammation after TBI. TIS restored GS clearance, enhanced OXA/OX2R-CaMKII signaling, strengthened dystrophin-associated AQP4 anchoring, re-established perivascular AQP4 polarization, and modulated astrocyte reactivity-associated markers. OX2R blockade markedly attenuated the effects of TIS on GS function, neuroinflammation, and behavioral outcomes. In vitro, OXA reduced C3 expression, increased S100A10 expression, and attenuated neuronal and endothelial injury in astrocyte co-cultures; these effects were diminished by AQP4 overexpression. Collectively, LH-targeted TIS improves GS function after TBI, at least partly through OXA/OX2R-CaMKII-associated regulation of dystrophin-associated AQP4 organization and polarization, thereby supporting astrocyte, neurovascular, and synaptic homeostasis and improving neurological outcomes.