To test whether silencing A20 could counteract the anti-necroptotic role of Nec-1 and melatonin, SD rats were randomly assigned into six groups: sham+AAV-shCtrl group (n = 25), CCI+AAV-shCtrl group (n = 27), CCI+Nec-1+AAV-shCtrl (n = 27), CCI+Melatonin+AAV-shCtrl (n = 27), CCI+Nec-1+AAV-shA20 (n = 27), CCI+Melatonin+AAV-shA20 (n = 27). Tissue samples were collected at 6 h except for behavioral test (Supplementary Figure S1D ). A schedule for AAV administration and drug treatment for experiment design 4 was displayed in Supplementary Figure S1E .
Melatonin (N-acetyl-5-methoxytryptamine, #M5250) was dissolved in 5% ethanol in saline (20 mg/kg) and injected into the left side of the peritoneum at 1 h before the CCI (Wong et al., 2014 (link)). Nec-1 (Selleck, Houston, TX, USA) and Z-VAD (BioVision, Mountain View, CA, USA) were dissolved in 10% dimethyl sulfoxide (Sigma-Aldrich). Rats was administered Nec-1 or Z-VAD via intracerebroventricular injection (coordinates: 0.8 mm posterior, 1.5 mm right lateral, and 4.0 mm ventral from Bregma). Infusion of 2 μg Z-VAD in 10 μl vehicle was conducted at a rate of 20 μl/h for 30 min (Li et al., 2014 (link)). Six microliter Nec-1 with concentration of 25 mM was used at a rate of 0.5 μl/min (Liu et al., 2016 (link)). In this study, experimenters made efforts to minimize the pain of rats.
Melatonin (N-acetyl-5-methoxytryptamine, #M5250) was dissolved in 5% ethanol in saline (20 mg/kg) and injected into the left side of the peritoneum at 1 h before the CCI (Wong et al., 2014 (link)). Nec-1 (Selleck, Houston, TX, USA) and Z-VAD (BioVision, Mountain View, CA, USA) were dissolved in 10% dimethyl sulfoxide (Sigma-Aldrich). Rats was administered Nec-1 or Z-VAD via intracerebroventricular injection (coordinates: 0.8 mm posterior, 1.5 mm right lateral, and 4.0 mm ventral from Bregma). Infusion of 2 μg Z-VAD in 10 μl vehicle was conducted at a rate of 20 μl/h for 30 min (Li et al., 2014 (link)). Six microliter Nec-1 with concentration of 25 mM was used at a rate of 0.5 μl/min (Liu et al., 2016 (link)). In this study, experimenters made efforts to minimize the pain of rats.
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