Background:Histopathology identified the anatomical and molecular abnormalities of brainstem nuclei in migraine patients. However, the exact whole brainstem structural changes in vivo have not yet been identified in medication-overuse headache (MOH) transformed from migraine. The aim of this study was to investigate the regional volume changes over the whole brainstem in the MOH patients using voxel-based morphometry (VBM) in vivo.Methods:High-resolution three-dimensional structural images were obtained using a 3.0-Tesla magnetic resonance system from 36 MOH patients and 32 normal controls (NCs) who were consecutively recruited from the International Headache Center, Chinese People's Liberation Army General Hospital, from March 2013 to June 2016. VBM was used to assess the brainstem structural alteration in the MOH patients, and voxel-wise correlation was performed to evaluate the relationship with the clinical characteristics.Results:The brainstem region with increased volume located in the left ventrolateral periaqueductal gray (MNI coordinate: -1, -33, -8), ventral tegmental area (MNI coordinate: 0, -22, -12), bilateral substantia nigra (MNI coordinate: -8, -16, -12, 9, -16, -12), and trigeminal root entry zone (MNI coordinate: -19, -29, -31; 19, -32, -29) in MOH patients compared with NCs. The headache visual analog scale score was positively related with the left rostral ventromedial medulla (RVM) (MNI coordinate: -1, -37, -56; cluster size: 20; r = 0.602) in the MOH patients.Conclusions:The regional volume gain of brainstem could underlie the neuromechanism of impaired ascending and descending pathway in the MOH patients, and the left RVM volume alteration could imply the impaired tolerance of nociceptive pain input and could be used to assess the headache disability in the MOH patients.