This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation (rTMS) on chronic neuropathic pain in rats.The behavior of rats with experimental chronic neuropathic pain was observed,and the expression of neuronal nitric oxide synthase (nNOS) in the ipsilateral dorsal root ganglions (DRGs) and the activation and proliferation of astrocytes in the ipsilateral spinal dorsal horn were detected.Thirty-two male Sprague-Dawley rats were randomly divided into four groups:sham-operated group,sham-rTMS group,1 Hz group and 20 Hz group (8 rats in each group).Chronic constriction nerve injury induced by sciatic nerve ligation was made to establish the models of the chronic neuropathic pain in rats except those in the sham-operated group.Then we applied different frequencies of rTMS to the primary motor cortex (M1) contralateral to the pain side once daily for 10 consecutive days.Pain behavior scores were observed before and after treatment.Western blot analysis was used to detect the expression of nNOS in ipsilateral L4-6 DRGs.Double immunofluorescent labeling for glial fibrillary acidic protein (GFAP) and 5-bromo-2-deoxyuridine (BrdU) was employed to observe the activation and proliferation of astrocytes in the ipsilateral L4-6 spinal dorsal horn.After rTMS treatment,the spontaneous pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS group (P＜0.05).Moreover,the brush-evoked pain behavior scores were significantly lower in the 20 Hz group than those in the sham-rTMS and 1 Hz group (P＜0.05),suggesting that the spontaneous pain and brush-evoked pain in the 20 Hz group were significantly alleviated.Western blot analysis revealed that the expression of nNOS in ipsilateral L4-6 DRGs was significantly decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P＜0.01) after rTMS treatment.Double immunofluorescence suggested that the expression of GFAP and the co-localization with BrdU in astrocytes were less in the sham-operated group than those in the sham-rTMS group and the 1 Hz group in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.After rTMS treatment,the expression of GFAP and the co-localization with BrdU decreased in the 20 Hz group as compared with the sham-rTMS group and the 1 Hz group (P＜0.05).In addition,the alleviation degree of spontaneous pain and brush-evoked pain in the 20 Hz group was negatively correlated with the expression of nNOS in ipsilateral DRGs and the number of GFAP/BrdU co-labelled astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain (P＜0.05).It was suggested that high-frequency rTMS may relieve neuropathic pain through down-regulating the overexpression of nNOS in ipsilateral DRGs and inhibiting the activity and proliferation of astrocytes in L4-6 spinal dorsal horn ipsilateral to the neuropathic pain.