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 4

In situ administration of Fer-1 reduces degenerating neurons and neurologic deficit in vivo.

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In situ administration of Fer-1 reduces degenerating neurons and neurolo...
(A and B) Male C57BL/6 mice (6–8 weeks old) underwent collagenase injection or sham procedure. Brain sections were stained with Fluoro-Jade B (FJB) at indicated times. (A) Field of interest and representative images of intracerebral hemorrhage (ICH) mouse brain. (B) Quantification of FJB+ cells. *P < 0.05 versus all other time points. Sham, 6 hours, 12 hours, 7 days, and 14 days: n = 8; 1 day: n = 10; 3 days: n = 12. (C–G) Mice were injected with collagenase followed by ferrostatin-1 (Fer-1) or vehicle in situ. Brains were collected and frozen sectioned at 1 and 3 days. (C) Sections were stained with FJB, and quantification is shown. **P < 0.01, ***P < 0.001 versus vehicle at the same time point. (D) Cresyl violet (CV) was used to stain surviving neurons at day 3. Representative images and quantification are shown. *P < 0.05 versus vehicle. (E) Perls’ staining and quantification of iron-positive cells. **P < 0.01 versus vehicle. (F) Brain sections were stained with CV and lesion volume calculated. **P < 0.01 versus vehicle. (G) Neurologic deficit score, right front paw placement, and hind limb placing scores. **P < 0.01 versus sham at the same time point; #P < 0.05 versus vehicle at the same time point. 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). One-way ANOVA followed by Dunn’s multiple comparison post test was used. For C–G, sham and vehicle: n = 8; ICH and Fer-1: n = 10. Scale bars: 50 μm (A and D), 100 μm (C and E), 1 mm (F). Original magnification of insets: ×400 (C), ×600 (D and E).