N-acetylcysteine effects on a murine model of chronic critical limb ischemia.

During chronic limb ischemia, oxidative damage and inflammation are described. Besides oxidative damage, the decrease of tissue oxygen levels is followed by several adaptive responses. The purpose of this study was to determine whether supplementation with N-acetylcysteine (NAC) is effective in an animal model of chronic limb ischemia. Chronic limb ischemia was induced and animals were treated once a day for 30 consecutive days with NAC (30mg/kg). After this time clinical scores were recorded and soleus muscle was isolated and lactate levels, oxidative damage and inflammatory parameters were determined. In addition, several mechanisms associated with hypoxia adaptation were measured (vascular endothelial growth factor - VEGF and hypoxia inducible factor - HIF levels, ex vivo oxygen consumption, markers of autophagy/mitophagy, and mitochondrial biogenesis). The adaptation to chronic ischemia in this model included an increase in muscle VEGF and HIF levels, and NAC was able to decrease VEGF, but not HIF levels. In addition, ex vivo oxygen consumption under hypoxia was increased in muscle from ischemic animals, and NAC was able to decrease this parameter. This effect was not mediated by a direct effect of NAC on oxygen consumption. Ischemia was followed by a significant increase in muscle myeloperoxidase activity, as well as interleukin-6 and thiobarbituric acid reactive substances species levels. Supplementation with NAC was able to attenuate inflammatory and oxidative damage parameters, and improve clinical scores. In conclusion, NAC treatment decreases oxidative damage and inflammation, and modulates oxygen consumption under hypoxic conditions in a model of chronic limb ischemia.

Ammonium tetrathiomolybdate following ischemia/reperfusion injury: Chemistry, pharmacology, and impact of a new class of sulfide donor in preclinical injury models.

BACKGROUND: Early revascularization of ischemic organs is key to improving outcomes, yet consequent reperfusion injury may be harmful. Reperfusion injury is largely attributed to excess mitochondrial production of reactive oxygen species (ROS). Sulfide inhibits mitochondria and reduces ROS production. Ammonium tetrathiomolybdate (ATTM), a copper chelator, releases sulfide in a controlled and novel manner, and may offer potential therapeutic utility. METHODS AND FINDINGS: In vitro, ATTM releases sulfide in a time-, pH-, temperature-, and thiol-dependent manner. Controlled sulfide release from ATTM reduces metabolism (measured as oxygen consumption) both in vivo in awake rats and ex vivo in skeletal muscle tissue, with a superior safety profile compared to standard sulfide generators. Given intravenously at reperfusion/resuscitation to rats, ATTM significantly reduced infarct size following either myocardial or cerebral ischemia, and conferred survival benefit following severe hemorrhage. Mechanistic studies (in vitro anoxia/reoxygenation) demonstrated a mitochondrial site of action (decreased MitoSOX fluorescence), where the majority of damaging ROS is produced. CONCLUSIONS: The inorganic thiometallate ATTM represents a new class of sulfide-releasing drugs. Our findings provide impetus for further investigation of this compound as a novel adjunct therapy for reperfusion injury.