Medial prefrontal cortex-targeted low-intensity focused ultrasound alleviates migraine-related allodynia and affective symptoms via modulating glutamate/GABA balance in mice

Migraine affects nearly one billion people worldwide and remains a major global health burden with inadequate treatment options. In this study, transcranial low-intensity focused ultrasound (LIFU) targeting the medial prefrontal cortex (mPFC) was assessed as a novel, non-invasive neuromodulatory strategy for migraine abortive treatment. The therapeutic potential and safety of LIFU were evaluated in animal models to support its future clinical translation. Male C57BL/6 mice were administered intraperitoneally with nitroglycerin (NTG) to induce an acute migraine model, whereas control animals received vehicle (VEH). Following model induction, mice were subjected to either mPFC-targeted LIFU or sham stimulation, which underwent an identical protocol without ultrasonic output. A series of behavioral, histological, and molecular assays was performed to evaluate the therapeutic effects and neuromodulatory mechanisms of LIFU. Mechanical allodynia was assessed using the von Frey test; anxiety-like behavior was evaluated in the elevated plus maze. Neuronal activation was examined via c-Fos immunofluorescence and GABA/glutamate co-staining. Tissue safety was assessed by HE staining and TUNEL assay. All quantitative analyses were conducted under blinded conditions. NTG injection induced significant allodynia and anxiety-like behaviors. LIFU stimulation significantly attenuated cephalic and plantar allodynia and ameliorated anxiety-like behaviors compared to the sham group. The NTG-induced migraine model exhibited significant mPFC c-Fos hyperactivation, which LIFU stimulation effectively suppressed. NTG injection significantly increased the proportion of activated glutamatergic neurons (GLU-N) and decreased activated GABAergic neurons (GABA-Ns) among total c-Fos-positive cells in the mPFC. This imbalance was reversed by LIFU, i.e., it was characterized by a decrease in activated GLU-Ns and an increase in activated GABA-Ns. No significant histopathological damage or apoptosis was detected following LIFU exposure. Aberrant activation and excitatory and inhibitory (E/I) imbalance of neurons in mPFC were involved in acute NTG-induced episodes. The LIFU targeting mPFC could alleviate NTG-induced mechanical allodynia and anxiety-like behavior by restoring E/I balance. LIFU is a novel, safe, non-invasive neuromodulatory strategy offering a potential migraine treatment.