# N-Acetylcysteine Reduces Tissue Injury Induced by Oxygen–Glucose Deprivation in an Organotypic Culture of Mouse Cerebral Cortex Slices

## TL;DR

N-acetylcysteine protects brain tissue from hypoxia in a lab model, with stronger effects in female mice, suggesting potential for treating newborn brain injury.

## Contribution

Demonstrates N-acetylcysteine's cytoprotective effect in an organotypic mouse cortex model and identifies optimal dosing and timing.

## Key findings

- N-acetylcysteine reduces hypoxia-ischemia-induced tissue damage in mouse cerebral cortex slices.
- Higher concentrations of N-acetylcysteine are more effective when administered before or shortly after hypoxia.
- Female mouse slices show greater protection and increased glutathione levels compared to male slices.

## Abstract

What are the main findings?
•In an in vitro model of hypoxic–ischemic cell death, N-acetylcysteine exerts a direct, concentration-dependent cytoprotective effect on cerebral cortex slices whether administered before or after oxygen–glucose deprivation.•N-acetylcysteine is more potent in reducing cellular damage in cerebral cortex slices derived from female mice than in those from male mice. This effect is associated with an increase in total glutathione levels in the tissue.

In an in vitro model of hypoxic–ischemic cell death, N-acetylcysteine exerts a direct, concentration-dependent cytoprotective effect on cerebral cortex slices whether administered before or after oxygen–glucose deprivation.

N-acetylcysteine is more potent in reducing cellular damage in cerebral cortex slices derived from female mice than in those from male mice. This effect is associated with an increase in total glutathione levels in the tissue.

What are the implications of the main findings?
•It is anticipated that systemic doses of N-acetylcysteine capable of achieving brain drug concentrations similar to those that are cytoprotective in vitro may be effective in in vivo models of hypoxia. This marks a further step toward the potential application of N-acetylcysteine to reduce hypoxia-induced brain damage in newborns.

It is anticipated that systemic doses of N-acetylcysteine capable of achieving brain drug concentrations similar to those that are cytoprotective in vitro may be effective in in vivo models of hypoxia. This marks a further step toward the potential application of N-acetylcysteine to reduce hypoxia-induced brain damage in newborns.

Background/Objectives: Hypoxic–ischemic encephalopathy is the leading cause of infant mortality and disability. Hypothermic therapy is effective in hypoxic–ischemic encephalopathy, albeit in a limited number of cases. Hypothermia requires advanced technologies and significant financial resources, which are difficult to sustain in low-income countries, with devastating consequences. Valid alternatives to hypothermia therapy are therefore needed. Methods: In vitro organotypic cultures of mouse cerebral cortex slices were used to demonstrate the direct protective effect of N-acetylcysteine (NAC) against brain tissue damage induced by oxygen–glucose deprivation (OGD), and to identify the concentrations and time window that maximize the drug’s effectiveness. NAC’s effectiveness was measured by the incorporation of propidium iodide (PI), a marker of cell membrane integrity. Results: Adding 0.1 and 1 mM NAC to the incubation medium before OGD strongly reduced OGD-induced PI incorporation, by 80% (p < 0.0002) and 89% (p < 0.0001), respectively. Administration of 1 mM NAC 1 h after OGD maintained a high degree of protection against OGD-induced damage (80% reduction in PI incorporation; p < 0.0001), while at 0.1 mM, the efficacy of NAC dropped to 44% (p < 0.005). Administration of NAC 4 h after OGD reduced PI incorporation to 52% (p < 0.005) at 1 mM, while at 0.1 mM, the effect was not significant (17%; p > 0.05). Exposure of slices to 0.1 and 1 mM NAC reduced PI incorporation in female cerebral cortex slices (p < 0.006), while only the higher concentration was effective in male slices (p < 0.05). Exposure to 0.1 mM NAC increased tissue levels of total glutathione (p = 0.0185), while no significant effect was observed with 1 mM NAC. Conclusions: This work highlights the direct effect of NAC in protecting cerebral cortex cells from OGD-induced damage and identifies the concentrations and time window that maximize the drug’s effect. The results underscore the need for further studies to verify the in vivo efficacy of NAC at concentrations found to be active in vitro, and for clinical trials to evaluate whether NAC can reduce hypoxia-induced brain damage in newborns.