Since NAC had this effect upon mitochondrial function it was
Since NAC had this effect upon mitochondrial function it was also determined muscle oxidative damage and inflammation. Guillot et al. (2014)  demonstrate in a rodent model of aortic cross-clamping that ROS are synthetized in a biphasic fashion, with a substantial increase during ischemia alone, followed by a normalization 10min after the beginning of reperfusion, and a secondary enhance 2h after reperfusion. Some authors demonstrated the effects of antioxidants vitamins in this model , , , , but the effects of NAC are not well known. Prolonged ischemia stimulates the excessive production of ROS, which may be potentiated by the reperfusion process, inducing tissue oxidative stress with various molecular damage, including changes in amino acids, lipids, membrane-carrying proteins and nucleic acids . ROS are known to be responsible, in part, for ischemic injury. Kahraman and colleagues (1997)  showed a significant increase in MDA production within 2h in a rabbit model of tourniquet-induced skeletal muscle ischemia-reperfusion injury. Acute skeletal muscle ischemia/reperfusion (I/R) is associated with increased levels of malondialdehyde, nitric oxide and protein carbonyl content, compared to the control group , and this is also observed after chronic ischemia. In addition, the revascularization of ischemic limbs is able to decrease oxidative damage parameters even in humans , and the administration of antioxidants can decrease oxidative damage after limb revascularization . In addition, hypoxia limits the supply of oxygen available to accept electrons during fatty AUY922 (NVP-AUY922) oxidation, which resulted in mitochondrial injury . Furthermore, NO is also involved in oxidative damage . During ischemia, NO can also be generated through non-enzymatic sources, such as the reduction of nitrite . Nitrosative and oxidative modifications generally constitute redox-related events that promote mitochondrial damage . On the other hand, NO-signaling increases peripheral blood flow in areas of tissue hypoxia . Besides oxidative damage, inflammatory response is also related to tissue damage after chronic ischemic injury. Actually, inflammation and oxidative damage seems to be related events during chronic limb ischemia . NAC significantly reduce both MPO activity and IL-6 levels, reinforcing the relation between oxidative stress and inflammation. Several other aspects of the adaptation of muscle to ischemia could be modified by NAC treatment, including the modulation of signaling pathways that improve vascularization or mitochondrial function / adaptation to hypoxia. Hypoxia-inducible factor 1α (HIF-1 α) is a transcriptional activator that functions as a regulator of oxygen homeostasis . It was previously demonstrated that NAC could modulate HIF expression in different models of ischemia/reperfusion injury  or chronic hypoxia . During limb ischemia HIF-1α is in its active form , as we demonstrate here, but NAC is not able to modulate it, suggesting that NAC effects were not secondary to HIF up-regulation. VEGF is an endothelial growth factor that acts on revascularization, maintaining the supply of oxygen and nutrients after chronic ischemia . Ischemia induces the production of angiogenic factors, which trigger sprouting of new capillaries (angiogenesis) and remodeling of preexisting vessels (arteriogenesis) . VEGF could also be related to tissue edema and inflammation , . VEGFR protein expression on the branched vasculature in ischemic legs increases gradually between day 4 and day 9 after hypoxic stimulation, peaks at approximately day 9, and then remains steady . We here demonstrated that NAC was able to decrease VEGF levels, and this is consistent with in vitro , ,  and in vivo ,  studies, but suggests that the protective effects of NAC are not related to the up-regulation of VEGF.