Figure 1.

N‐acetylcysteine (NAC) abrogated hemin‐induced ferroptosis in primary neurons in vitro and reduced cell death and enhanced functional recovery in a collagenase ICH model in mice. (A) Representative images of primary cortical neurons 24 hours after treatment with saline, NAC (1mM), hemin (100 μM) and hemin (100 μM) + NAC (1mM). LIVE/DEAD assay; green fluorescent cells labeled with calcein‐AM are alive; red fluorescent cells labeled with ethidium homodimer are dead. Scale bars, 100 μm. (B) NAC protected primary cortical neurons from hemin‐induced ferroptosis in a concentration‐dependent manner. Cell death was analyzed 24 hours after hemin treatment with or without NAC by monitoring MTT reduction, a population measure of cell viability. (C) Experimental design for delivery of NAC post‐ICH in mice. NAC (75mg and 300mg/kg; intraperitoneal) was delivered 2 hours after collagenase injection and then daily for 7 days. Behavior was assessed using the corner task (spatial neglect) and adhesive tape removal task (sensory neglect) and was assessed on days 1, 3, and 7 after ICH. (D) NAC reduced neuronal degeneration was monitored by Fluoro‐Jade (FJ) staining (green) in the perihematomal regions of the mouse brain. Representative images show increased numbers of degenerating neurons (white arrows) in the ICH‐treated group; this was reduced by NAC treatment. Scale bar, 100 μm. (E) Quantification of FJ staining of neurons, a nonspecific marker of degeneration, after NAC treatment in ICH mice brains. (F) NAC (300mg/kg) significantly reduces spatial neglect associated with ICH. (G) NAC (300mg/kg) reduces sensory neglect (adhesive tape removal task) induced by ICH (n = 11). NAC (75mg/kg) had no significant effect in mice or rats (not shown). Significance was determined by two‐way ANOVA and Bonferroni's post‐hoc test. All graphs are mean ± SEM. ANOVA = analysis of variance; ICH = intracerebral hemorrhage; NAC = N‐acetylcysteine; MTT = methyl thiazolyl tetrazolium; SEM = standard error of the mean.