Simple SummaryChronic inflammatory diseases could impact central nervous system homeostasis, being oxidative damage of the dorsal horn, a relevant mechanism mediating central sensitization. Chronic inflammatory lameness in dairy cows is a painful condition that affects animal welfare, affecting dairy production worldwide. This study reveals increased levels of reactive oxygen species, malondialdehyde, and carbonyl groups, indicating lipid and protein damage in the spinal cord of cows with chronic lameness. Moreover, antioxidant system activity was similar between lame and non-lame cows which suggests that antioxidant dysregulation was not the cause of oxidative damage, as has been proposed previously. Based on the fact that nociceptive pathways are strongly conserved between species, there is no reason to neglect that chronic pain in cows promotes Central Nervous System (CNS) alterations, such as oxidative damage. Moreover, lame cows develop central sensitization, as allodynia and hyperalgesia are centrally and not peripherally mediated. Our results support the current assumption that chronic pain is a central nervous system disease and lameness in dairy cows is far beyond an inflammation of the hoof.AbstractLameness in dairy cows is a worldwide prevalent disease with a negative impact on animal welfare and herd economy. Oxidative damage and antioxidant system dysfunction are common features of many CNS diseases, including chronic pain. The aim of this study was to evaluate the levels of reactive oxygen species (ROS) and oxidative damage markers in the spinal cord of dairy cows with chronic inflammatory lameness. Locomotion score was performed in order to select cows with chronic lameness. Dorsal horn spinal cord samples were obtained post mortem from lumbar segments (L2–L5), and ROS, malondialdehyde (MDA), and carbonyl groups were measured along with the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant response (TAR). Lame cows had increased levels of ROS, MDA, and carbonyl groups, while no differences were observed between lame and non-lame cows in SOD, GPx, CAT, and TAR activity. We conclude that painful chronic inflammatory lameness in dairy cows is associated with an increase in ROS, MDA, and carbonyl groups. Nonetheless, an association between ROS generation and dysfunction of the antioxidant system, as previously proposed, could not be established.