Cardiac arrest and CPR increased the percentage of Fluoro-Jade B positive cells to DAPI-positive cells in the cortex, caudoputamen, and hippocampus in WT mice

Cardiac arrest and CPR increased the percentage of Fluoro-Jade B positive cells to DAPI-positive cells in the cortex, caudoputamen, and hippocampus in WT mice. CA/CPR. Administration of SPL-334.1 attenuated the increase in GSNOR activity in brain, heart, liver, spleen, and plasma, and restored S-nitrosylated protein levels in the brain. Inhibition of GSNOR attenuated ischemic brain injury and improved survival in WT mice after CA/CPR (81.8% in SPL-334.1 vs. 36.4% in placebo, Log Rank P=0.031). Similarly, GSNOR deletion prevented the reduction in the number of S-nitrosylated proteins in the brain, mitigated brain injury, and improved neurological recovery and survival after CA/CPR. Both TOK-8801 GSNOR inhibition and deletion attenuated CA/CPR-induced disruption of blood brain barrier. Post-cardiac arrest patients experienced higher plasma GSNOR activity than did pre-operative cardiac surgery patients or healthy volunteers (P<0.0001). Plasma GSNOR activity was positively correlated with initial lactate levels in post-arrest patients (Spearman rs=0.48, P=0.045). Conclusions: Cardiac arrest Rabbit polyclonal to ACTR5 and CPR activated GSNOR and reduced the number of S-nitrosylated proteins in the brain. Pharmacological inhibition or genetic deletion of GSNOR prevented ischemic brain injury and improved survival rates by restoring S-nitrosylated protein levels in the brain after CA/CPR in mice. Our observations suggest that GSNOR is usually a novel biomarker of post-arrest brain injury as well as a molecular target to improve outcomes after cardiac arrest. use as a sodium salt. WT mice were subjected to CA and received either SPL-334.1 (6 mg/kg) or the same volume TOK-8801 (100 L) of normal saline (placebo) intravenously at 15 min after ROSC in a randomized and blinded manner. SPL-334.1 was dissolved in sterile distilled water, pH 8.0, adjusted with 1M NaHCO3. Because we found that mice treated with normal saline or distilled TOK-8801 water had similar survival rates and inflammatory response after CA/CPR in pilot experiments, we used normal saline as placebo. To minimize variability, we used randomized paired (a.k.a., matched pairs) design.11 We paired inbred C57BL/6 mice to SPL-334.1 or placebo on the basis of similar weight, age, delivery date, and when possible holding cage. Pairing appeared effective (Supplemental Table 1). Sham-operated mice received anesthesia, surgery, and ventilation but no cardiac arrest and the same post-resuscitation care until the similarly timed endpoint. Sham-operated mice were drawn from your same batch as experimental animals in all experiments except for plasma cytokine measurements. Plasma GSNOR activity was measured in post-CA GSNOR?/? mice as unfavorable control in an unblinded manner. Measurements of GSNOR activity We measured GSNOR activity at 6 hours after CA/CPR based on pilot experiments (data not shown). The rate of GSNO-dependent NADH consumption was measured to assess GSNOR activity levels in plasma and tissue homogenates, as previously described.20,30 Samples were homogenized in a buffer containing (in mmol/l) 50 Tris-HCl (pH 8.0), 150 NaCl, 1 EDTA, 0.1% Triton X-100, and 1:100 protease inhibitor cocktail. After centrifugation for 10 min at 10,000 g, samples were diluted to a protein concentration of 0.1 mg/ml or a plasma concentration of 1 1.0 mg/ml in reaction buffer containing 20 mmol/l Tris-HCl (pH 8.0), 0.5 mmol/l EDTA, and incubated with 75 mol/l NADH with or without 100 mol/l GSNO. NADH consumption was monitored by fluorescence spectrophotometry with excitation at 340 nm and emission at 460 nm. GSNOR activity was defined as the rate of NADH consumption in samples incubated in the presence TOK-8801 of GSNO minus the rate of NADH consumption without GSNO. Assessment of Neurological Function Neurological function score (NFS) was assessed at 96 hours after CA/CPR by an investigator blinded to the experimental group using a previously-reported neurological function scoring system with minor modifications.6,7,28,29 Dead or brain death mice were scored at 0 points. Dead mice were excluded from your statistical analysis. S-nitrosylated protein and peptide TOK-8801 identification after CA/CPR Total S-nitrosylated proteins were detected by SNO-RAC (resin-assisted capture of S-nitrosylated proteins) as previously explained.31 Label-free peptide identification and quantification was then performed with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system (LTQ Orbitrap XL mass spectrometer, Thermo Fisher Scientific) at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School). Peptide and protein identifications were extracted from your Sequest database using default settings. Peptides were counted if they were recovered in any single sample, and were counted only once per experimental group. Proteins were considered present if 2 or more.