Association between hippocampal ferroptosis-related changes and behavioral alterations in CCI rats with neuropathic pain

This study aimed to characterize behavioral alterations induced by chronic constriction injury (CCI) of the sciatic nerve and hippocampal ferroptosis in rats and to explore the association between hippocampal ferroptosis and behavioral changes accompanying neuropathic pain (NP). Ferrostatin-1 (10 mg/kg) and Erastin (10 mg/kg) were injected intraperitoneally into thirty-two male SD rats for two consecutive days. Mechanical paw withdrawal threshold (MWT), paw withdrawal latency (PWL), and open field test (OFT) were measured before the operation and on days 7 and 14 after an operation, respectively.

Western Blot was used to detect GPX4, xCT, and FTH1, while kits were used to detect MDA and GSH expression. HE, Nissl, and immunofluorescence staining were also carried out to examine the tissues. Additionally, structural changes in the mitochondria were observed using a transmission electron microscope.

The MWT and PWL values in the CCI group were significantly lower than those in the Sham group on the 7th day after operation. Compared to the CCI group, CCI + F group showed significant increases in MWT and PWL, while PWL significantly decreased in the CCI + E group. Moreover, the CCI group showed a significant decrease in the total distance traveled and the number of central-area crossings in the OFT, while immobility time increased significantly; however, because overall locomotor activity was markedly reduced, the decrease in central exploration was not considered specific evidence of anxiety-like behavior.

The CCI + F group showed increased activity and less immobility compared to the CCI group. The CCI group of rats showed significant decreases in GPX4, xCT, FTH1 and GSH, while increase in MDA. CCI + F group had higher levels of GPX4, xCT, and FTH1 and GSH, while lower level of MDA compared to the CCI group. xCT and FTH1 levels decreased in the CCI + E group.

Furthermore, the CCI group’s hippocampal neurons suffered reduced Nissl bodies, lower c-Fos levels, and significantly damaged mitochondrial cristae. CCI + F hippocampal neurons exhibited, increased Nissl bodies and c-Fos compared to CCI. Hippocampal ferroptosis was associated with neuropathic pain and alterations in open-field behavior in CCI rats.

Inhibition of ferroptosis ameliorated pain sensitivity and partially reversed the CCI-induced behavioral alterations; however, the present OFT findings alone do not establish a specific causal relationship between hippocampal ferroptosis and anxiety-like behavior. Guangxi Medical Health Appropriate Technology Development and Promotion Application Project (No.S2023044); Guangxi Clinical Research Center for Anesthesiology (GK AD22035214); Shanghai Science and Technology Commission Medical Innovation Research Special Project (No. 20Y21903100); Shanghai Shenkang Hospital Development Center three-year Action Plan for Promoting Clinical Skills and Clinical Innovation in Municipal Hospitals (SHDC2020CR3102B); Budget-supported Project of Shanghai University of Traditional Chinese Medicine (No.2020LK063); Shanghai Traditional Chinese Medicine Hospital Future Plan Science and Technology Development Project (No.WL-HBMS-2022002 K). Xiaoxia Wang and Jianqiu Zheng contributed equally to this work.