Objectives N‐acetylcysteine (NAC) is a clinically approved thiol‐containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an “antioxidant,” poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke. Methods Hemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5‐lipoxygenase (ALOX5) knockout mice, and viral‐gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC. Results NAC prevented hemin‐induced ferroptosis by neutralizing toxic lipids generated by arachidonate‐dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione‐dependent enzymes such as glutathione S ‐transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E 2 (PGE 2 ). Interpretation NAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE 2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854–872

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Intracerebral hemorrhage (ICH) causes blood-brain barrier (BBB) damage along with altered element levels in the brain. BBB permeability was quantified at 3, 7, and 14 days with Evans Blue dye after collagenase-induced ICH in rat. At peak permeability (day 3), a gadolinium (Gd)-based contrast agent was injected to further characterize BBB disruption, and X-ray fluorescence imaging (XFI) was used to map Gd, Fe, Cl, and other elements. XFI revealed that Ca, Cl, Gd, and Fe concentrations were significantly elevated, whereas K was significantly decreased. Therefore, using Gd-XFI, we co-determined BBB dysfunction with alterations in the metallome, including those that contribute to cell death and functional outcome. Warfarin was administered 3 days post-ICH to investigate whether additional or new bleeding occurs during peak BBB dysfunction, and hematoma volume was assessed on day 4. Warfarin administration prolonged bleeding time after a peripheral cut-induced bleed, but warfarin did not worsen hematoma volume. Accordingly, extensive BBB leakage occurred after ICH, but did not appear to affect total hematoma size.

After intracerebral hemorrhage (ICH), brain edema commonly occurs and can cause death. Along with edema, there are significant alterations in the concentrations of key ions such as sodium, potassium, and chloride, which are essential to brain function. NKCC1, a cation-chloride cotransporter, is upregulated after brain damage, such as traumatic injury and ischemic stroke. NKCC1 brings sodium and chloride into the cell, possibly worsening ion dyshomeostasis. Bumetanide, a specific NKCC1 antagonist, blocks the transport of chloride into cells, and thus should attenuate the increases in chloride, which should lessen brain edema and improve neuronal functioning post-ICH, as with other injuries. We used the collagenase model of ICH to test whether bumetanide treatment for three days (vs. vehicle) would improve outcome. We gave bumetanide beginning at two hours or seven days post-ICH and measured behavioural outcome, edema, and brain ion content after treatment. There was some evidence for a minor reduction in edema after early dosing, but this did not improve behaviour or lessen injury. Contrary to our hypothesis, bumetanide did not normalize ion concentrations after late dosing. Bumetanide did not improve behavioural outcome or affect lesion volume. After ICH, bumetanide is safe to use in rats but does not improve functional outcome in the majority of animals.

Extensive animal research facilitated the clinical translation of therapeutic hypothermia for cardiac arrest in adults and hypoxic-ischemic injury in infants. Similarly, clinical interest in hypothermia for other brain injuries, such as stroke, has been greatly supported by positive findings in preclinical work. The reliability, validity, and utility of animal models, among many research practices (blinding, randomization, etc.), are key to successful clinical translation. Here, we review methods used to induce and maintain hypothermia in animal models. These include physical and pharmacological methods. We emphasize the advantages and limitations of each approach, and the importance of using clinically relevant cooling protocols and appropriate monitoring and reporting approaches. Moreover, we performed a literature survey of ischemic stroke studies published in 2015 to highlight the continuing risk of temperature confounds in neuroprotection studies. For example, many still do not accurately monitor and report temperature during surgery (23.5%), even though almost half of these studies (46.0%) use pharmaceutical agents that likely influence temperature. We hope this review stimulates awareness and discussion of the importance of temperature in neuroprotective studies.

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