Nuclear factor erythroid 2-related factor 2 and choline acetyltransferase co-expression in rat spinal cord neurons after ischemia-reperfusion injury.

Spinal cord ischemia-reperfusion injury (IRI) results in overproduction of reactive oxygen species leading to tissue oxidative stress which impacts the neuronal network in the spinal cord as well as glial cells. We investigated the expression of Nuclear factor erythroid 2-related factor 2 (Nrf2) in neurons and glial cells after occlusion of the abdominal aorta followed by IRI as well as the time-dependent expression of Nrf2 in the same cells. The experimental method of transient aortic occlusion was carried out on rats by cross-clamping of the abdominal aorta for 45 minutes. The animals used for this study were sacrificed 1 h, 6 h, and 48 h after reperfusion to determine time-related changes of Nrf2 expression, as well as changes of astrocyte activity in the spinal cord. Immunofluorescence results showed an increase in the staining intensity of Nrf2 expression in the neurons following ischemia with highest intensity 48 h post-reperfusion and an increase in a number of reactive astrocytes. Western blot analysis showed that Nrf2 protein expression increased in a cytoplasmic and nuclear fraction as early as 1 h after reperfusion and remained active 48 h after, resulting in increased expression of the main Nrf2 target gene HO-1. In conclusion, substances that enhance expression of Nrf2 may have the potential to prevent cellular damage to the spinal cord caused by IRI.

Mn porphyrin-based SOD mimic, MnTnHex-2-PyP(5+), and non-SOD mimic, MnTBAP(3-), suppressed rat spinal cord ischemia/reperfusion injury via NF-κB pathways.

Herein we have demonstrated that both superoxide dismutase (SOD) mimic, cationic Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP(5+)), and non-SOD mimic, anionic Mn(III) meso-tetrakis(4-carboxylatophenyl)porphyrin (MnTBAP(3-)), protect against oxidative stress caused by spinal cord ischemia/reperfusion via suppression of nuclear factor kappa B (NF-κB) pro-inflammatory pathways. Earlier reports showed that Mn(III) N-alkylpyridylporphyrins were able to prevent the DNA binding of NF-κB in an aqueous system, whereas MnTBAP(3-) was not. Here, for the first time, in a complex in vivo system-animal model of spinal cord injury-a similar impact of MnTBAP(3-), at a dose identical to that of MnTnHex-2-PyP(5+), was demonstrated in NF-κB downregulation. Rats were treated subcutaneously at 1.5 mg/kg starting at 30 min before ischemia/reperfusion, and then every 12 h afterward for either 48 h or 7 days. The anti-inflammatory effects of both Mn porphyrins (MnPs) were demonstrated in the spinal cord tissue at both 48 h and 7 days. The downregulation of NF-κB, a major pro-inflammatory signaling protein regulating astrocyte activation, was detected and found to correlate well with the suppression of astrogliosis (as glial fibrillary acidic protein) by both MnPs. The markers of oxidative stress, lipid peroxidation and protein carbonyl formation, were significantly reduced by MnPs. The favorable impact of both MnPs on motor neurons (Tarlov score and inclined plane test) was assessed. No major changes in glutathione peroxidase- and SOD-like activities were demonstrated, which implies that none of the MnPs acted as SOD mimic. Increasing amount of data on the reactivity of MnTBAP(3-) with reactive nitrogen species (RNS) (.NO/HNO/ONOO(-)) suggests that RNS/MnTBAP(3-)-driven modification of NF-κB protein cysteines may be involved in its therapeutic effects. This differs from the therapeutic efficacy of MnTnHex-2-PyP(5+) which presumably occurs via reactive oxygen species and relates to NF-κB thiol oxidation; the role of RNS cannot be excluded.