Autophagy may contribute to ischemia-induced cell death in the brain, but the regulation of autophagic cell death is largely unknown. Nuclear factor kappa B (NF-κB) is a regulator of apoptosis in cerebral ischemia. We examined the hypothesis that autophagy-like cell death could contribute to ischemia-induced brain damage and the process was regulated by NF-κB. In adult wild-type (WT) and NF-κB p50 knockout (p50−/−) mice, focal ischemia in the barrel cortex was induced by ligation of distal branches of the middle cerebral artery. Twelve to 24 h later, autophagic activity increased as indicated by enhanced expression of Beclin-1 and LC3 in the ischemic core and/or penumbra regions. This increased autophagy contributed to cell injury, evidenced by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) co-staining and a protective effect achieved by the autophagy inhibitor 3-methyladenine. The number of Beclin-1/TUNEL-positive cells was significantly more in p50−/− mice than in WT mice. Neuronal and vascular cell death, as determined by TUNEL-positive cells co-staining with NeuN or Collagen IV, was more abundant in p50−/− mice. Immunostaining of the endothelial cell tight junction marker occludin revealed more damage to the blood–brain barrier in p50−/− mice. Western blotting of the peri-infarct tissue showed a reduction of Akt-the mammalian target of rapamycin (mTOR) signaling in p50−/− mice after ischemia. These findings provide the first evidence that cerebral ischemia induced autophagy-like injury is regulated by the NF-κB pathway, which may suggest potential treatments for ischemic stroke.

Autophagy is upregulated in spinal cord ischemia reperfusion (SCIR) injury; however, its expression mechanism is largely unknown; moreover, whether autophagy plays a neuroprotective or neurodegenerative role in SCIR injury remains controversial. To explore these issues, we created an SCIR injury rat model via aortic arch occlusion. Compared with normal controls, autophagic cell death was upregulated in neurons after SCIR injury. We found that autophagy promoted neuronal cell death during SCIR, shown by a significant number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling- (TUNEL-) positive cells colabeled with the autophagy marker microtubule-associated protein 1 light chain 3, while the autophagy inhibitor 3-methyladenine reduced the number of TUNEL-positive cells and restored neurological and motor function. Additionally, we showed that oxidative stress was the main trigger of autophagic neuronal cell death after SCIR injury and N-acetylcysteine inhibited autophagic cell death and restored neurological and motor function in SCIR injury. Finally, we found that hydrogen sulfide (H2S) inhibited autophagic cell death significantly by reducing oxidative stress in SCIR injury via the AKT-the mammalian target of rapamycin (mTOR) pathway. These findings reveal that oxidative stress induces autophagic cell death and that H2S plays a neuroprotective role by reducing oxidative stress in SCIR.

The role of autophagy, neuroprotective mechanisms of nicotinamide adenine dinucleotide (NAD+), and their relationship in spinal cord ischemic reperfusion injury (SCIR) was assessed. Forty-eight Sprague-Dawley rats were divided into four groups: sham, ischemia reperfusion (I/R), 10 mg/kg NAD+, and 75 mg/kg NAD+. Western blotting, immunofluorescence, and immunohistochemistry were used to assess autophagy and apoptosis. Basso, Beattie, and Bresnahan (BBB) scores were used to assess neurological function. Expression levels of Beclin-1, Atg12-Atg5, LC3B-II, cleaved caspase 3, and Bax were upregulated in the I/R group and downregulated in the 75 mg/kg NAD+ group; p-mTOR, p-AKT, p62, and Bcl-2 were downregulated in the I/R group and upregulated in the 75 mg/kg NAD+ group. Numbers of LC3B-positive, caspase 3-positive, Bax-positive, and TUNEL-positive cells were significantly increased in the I/R group and decreased in the 75 mg/kg NAD+ group. The mean integrated option density of Bax increased and that of Nissl decreased in the I/R group, and it decreased and increased, respectively, in the 75 mg/kg NAD+ group. BBB scores significantly increased in the 75 mg/kg NAD+ group relative to the I/R group. No difference was observed between I/R and 10 mg/kg NAD+ groups for these indicators. Therefore, excessive and sustained autophagy aggravates SCIR; administration of NAD+ alleviates injury.