Inhibition of neuronal ferroptosis protects hemorrhagic brain
Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang
Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang
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Research Article Neuroscience

Inhibition of neuronal ferroptosis protects hemorrhagic brain

Abstract

Intracerebral hemorrhage (ICH) causes high mortality and morbidity, but our knowledge of post-ICH neuronal death and related mechanisms is limited. In this study, we first demonstrated that ferroptosis, a newly identified form of cell death, occurs in the collagenase-induced ICH model in mice. We found that administration of ferrostatin-1, a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin in organotypic hippocampal slice cultures (OHSCs). Mice treated with ferrostatin-1 after ICH exhibited marked brain protection and improved neurologic function. Additionally, we found that ferrostatin-1 reduced lipid reactive oxygen species production and attenuated the increased expression level of PTGS2 and its gene product cyclooxygenase-2 ex vivo and in vivo. Moreover, ferrostatin-1 in combination with other inhibitors that target different forms of cell death prevented hemoglobin-induced cell death in OHSCs and human induced pluripotent stem cell–derived neurons better than any inhibitor alone. These results indicate that ferroptosis contributes to neuronal death after ICH, that administration of ferrostatin-1 protects hemorrhagic brain, and that cyclooxygenase-2 could be a biomarker of ferroptosis. The insights gained from this study will advance our knowledge of the post-ICH cell death cascade and be essential for future preclinical studies.

Authors

Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang

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Figure 5

Intracerebroventricular administration of Fer-1 reduces degenerating neurons, neurologic deficit, and lipid ROS and inhibits COX-2 expression in vivo.

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Intracerebroventricular administration of Fer-1 reduces degenerating neu...
Male C57BL/6 mice (6–8 weeks old) underwent collagenase injection or sham procedure. Ferrostatin-1 (Fer-1) or vehicle was injected through the left ventricle 2 hours after collagenase. (A) Brain slices were stained with Fluoro-Jade B (FJB), and quantification is shown. *P < 0.05, **P < 0.01 versus corresponding vehicle. (B) Brain slices were stained with Cresyl violet (CV), and stereological quantification of the surviving neurons in the ipsilateral striatum is shown. ***P < 0.001 versus sham; #P < 0.05 versus vehicle. (C) Lesion volume was calculated after CV/Luxol fast blue staining. *P < 0.05 versus corresponding vehicle. Scale bar: 1 mm. (D) Neurologic deficit score, right front paw placement, and hind limb placing scores. *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding sham; #P < 0.05 versus corresponding vehicle. (E–G) Four-millimeter tissue slices were collected from the hematoma core and perihematoma region of intracerebral hemorrhage (ICH) and sham animals. Tissue was homogenized for the malondialdehyde (MDA) assay (E) and Western blotting (F and G). β-Actin served as a loading control. Protein expression was normalized to β-actin and expressed as fold change of sham. *P < 0.05, ***P < 0.001 versus sham; #P < 0.05, ##P < 0.01, ###P < 0.001 versus corresponding vehicle. Results are shown as box-and-whisker plots (the middle horizontal line within the box represents the median, boxes extend from the 25th to the 75th percentile, and the whiskers represent 95% confidence intervals). Statistical tests used were 1-way ANOVA followed by Dunn’s multiple comparison post test (A–D) and 1-way ANOVA followed by Bonferroni post hoc analysis (D–F). For A–D, sham and vehicle: n = 8; ICH and Fer-1: n = 10. For E–G, sham, vehicle, and Fer-1: n = 5.