A modified FLIT-B model of trigeminal neuralgia reveals anterior cingulate cortex involvement in anxiety-like behavior in mice

BACKGROUND: Trigeminal neuralgia (TN) is a debilitating orofacial pain disorder characterized by paroxysmal, lancinating pain and prominent affective comorbidities. Increasing evidence suggests that chronic TN involves abnormal plasticity in supraspinal pain circuits. However, the lack of animal models that faithfully recapitulate key clinical features of TN has limited mechanistic investigations of these central processes. Here, building upon our previously established foramen lacerum impingement of the trigeminal nerve root (FLIT) model, we developed an improved glass bead-based version (FLIT-B) that enables reproducible and controlled compression of the trigeminal nerve root and facilitates investigation of supraspinal mechanisms in TN. METHODS: In the FLIT-B model, calibrated glass beads were implanted through the foramen lacerum to compress the trigeminal nerve root. Behavioral assays were used to assess sensory and affective phenotypes, while histological analyses evaluated trigeminal nerve pathology. To examine supraspinal circuit alterations, neuronal activation in the primary somatosensory barrel field (S1BF), anterior cingulate cortex (ACC), amygdala, and hippocampus was assessed using c-Fos immunostaining. Structural and functional changes in excitatory neurons, particularly in the ACC, were further analyzed using AAV-mediated labeling, in vivo fiber photometry, and two-photon calcium imaging in both sham and FLIT-B mice. RESULTS: FLIT-B mice exhibited spontaneous pain-like behaviors, including asymmetric facial grimacing and peri-orbital hair loss associated with excessive grooming, suggesting persistent orofacial discomfort. Histological analyses confirmed consistent trigeminal nerve root demyelination. c-Fos staining and calcium imaging revealed widespread activation of the S1BF, ACC, amygdala, and hippocampus, with the ACC showing the highest degree of activation, particularly in spontaneous activity patterns. Morphological analyses demonstrated significant dendritic remodeling in excitatory neurons of the ACC and S1BF. Chemogenetic inhibition of ACC excitatory neurons significantly alleviated both pain-like and anxiety-like behaviors. CONCLUSIONS: The FLIT-B model provides a reproducible and clinically relevant rodent model of TN by compression of the trigeminal nerve root at an anatomically relevant site. Using this model, we identify ACC-centered structural and functional plasticity as a key supraspinal substrate linking chronic trigeminal pain to affective disturbances.