Ity was measured by recording time to complete Trailmaking A and

Ity was measured by recording time to complete Trailmaking A and B. Digit Span was used to measure working memory and sustained attention span. Fluency was assessed by requiring patients to generate words in a category or beginning with a specific letter. The Wechsler Test of Adult Reading was used as a test of premorbid intelligence. The Stroop Color-Word Inference Test was used to assess executive function, and the Visual Search and Acuity Test assessed visuospatial abilities and executive function. Patients with NCD were defined as those who demonstrated a 1-standard deviation decline from baseline on 25% of the tasks, in accordance with the “Statement of consensus on assessment of neurobehavioral outcomes after cardiac surgery”10. Sample Collection and Microarray Processing For all 42 patients, blood samples were collected from a central venous line preoperatively after induction of anesthesia and before skin incision, early or 6 hours postCPB in the intensive care unit, and late or 4 days post-CPB. Blood was drawn directly into PAXgene tubes for mRNA stabilization PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847069 and extraction, per the manufacturer’s recommendation. Skeletal muscle samples were collected from twenty patients from the left intercostal muscle bed after cannulation but before the initiation of CPB, and again after removal of the aortic cross clamp and weaning from CPB. Skeletal muscle samples were snap frozen in liquid Neuromedin N web nitrogen immediately after collection and stored at -80 C. RNA extraction and purification, cDNA synthesis, and production of biotin-labeled cRNA were completed by the Beth Isreal Deaconess Medical Center Proteomics Core according to previously described protocols11, 12. cRNA from all samples were hybridized with Affymetrix GeneChip HG-U133 Plus 2.0, which probes for over 38,500 genes. Chips were scanned with an HP G2500A ChipScanner, and low-level quality control analysis and signal value measurement was performed using dChip software 13. No outliers were identified by dChip, so all samples were carried on for subsequent analysis. Gene Expression and Pathway Analysis Gene expression analysis was performed on raw microchip data using JMP Genomics 4.0 for quality control, normalization, and statistical analysis. Composite chip data were normalized and compared using the Robust Multichip Average method, which revealed one blood and one skeletal muscle sample to be outliers. These were excluded from subsequent analysis. Gene expression in Pre-CPB and Post-CPB skeletal muscle samples and Pre-CPB, 6H Post-CPB, and 4D Post-CPB blood samples in patients with NCD were compared to the corresponding samples in patients without NCD using one-way ANOVA. A post-hoc false detection rate algorithm with alpha of 0.05 was applied to control for false Author Manuscript Author Manuscript Author Manuscript Author Manuscript J Thorac EMA401 site Cardiovasc Surg. Author manuscript; available in PMC 2016 February 01. Sabe et al. Page 5 positives. Genes that were considered significantly regulated met two criteria: 1) mean fold change >1.5 or <-1.5 in NCD patients compared to NORM, and 2) -log exceeding threshold calculated by the software for each comparison. All significant genes were uploaded into Ingenuity Pathway Analysis which was used to generate the top canonical pathways involving the differentially regulated genes. Real-time PCR Gene expression analysis of whole blood-derived mRNA with HGU 133 Plus 2.0 chips was previously validated by real-time PCR8. We used real-time PCR to.Ity was measured by recording time to complete Trailmaking A and B. Digit Span was used to measure working memory and sustained attention span. Fluency was assessed by requiring patients to generate words in a category or beginning with a specific letter. The Wechsler Test of Adult Reading was used as a test of premorbid intelligence. The Stroop Color-Word Inference Test was used to assess executive function, and the Visual Search and Acuity Test assessed visuospatial abilities and executive function. Patients with NCD were defined as those who demonstrated a 1-standard deviation decline from baseline on 25% of the tasks, in accordance with the "Statement of consensus on assessment of neurobehavioral outcomes after cardiac surgery"10. Sample Collection and Microarray Processing For all 42 patients, blood samples were collected from a central venous line preoperatively after induction of anesthesia and before skin incision, early or 6 hours postCPB in the intensive care unit, and late or 4 days post-CPB. Blood was drawn directly into PAXgene tubes for mRNA stabilization PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847069 and extraction, per the manufacturer’s recommendation. Skeletal muscle samples were collected from twenty patients from the left intercostal muscle bed after cannulation but before the initiation of CPB, and again after removal of the aortic cross clamp and weaning from CPB. Skeletal muscle samples were snap frozen in liquid nitrogen immediately after collection and stored at -80 C. RNA extraction and purification, cDNA synthesis, and production of biotin-labeled cRNA were completed by the Beth Isreal Deaconess Medical Center Proteomics Core according to previously described protocols11, 12. cRNA from all samples were hybridized with Affymetrix GeneChip HG-U133 Plus 2.0, which probes for over 38,500 genes. Chips were scanned with an HP G2500A ChipScanner, and low-level quality control analysis and signal value measurement was performed using dChip software 13. No outliers were identified by dChip, so all samples were carried on for subsequent analysis. Gene Expression and Pathway Analysis Gene expression analysis was performed on raw microchip data using JMP Genomics 4.0 for quality control, normalization, and statistical analysis. Composite chip data were normalized and compared using the Robust Multichip Average method, which revealed one blood and one skeletal muscle sample to be outliers. These were excluded from subsequent analysis. Gene expression in Pre-CPB and Post-CPB skeletal muscle samples and Pre-CPB, 6H Post-CPB, and 4D Post-CPB blood samples in patients with NCD were compared to the corresponding samples in patients without NCD using one-way ANOVA. A post-hoc false detection rate algorithm with alpha of 0.05 was applied to control for false Author Manuscript Author Manuscript Author Manuscript Author Manuscript J Thorac Cardiovasc Surg. Author manuscript; available in PMC 2016 February 01. Sabe et al. Page 5 positives. Genes that were considered significantly regulated met two criteria: 1) mean fold change >1.5 or <-1.5 in NCD patients compared to NORM, and 2) -log exceeding threshold calculated by the software for each comparison. All significant genes were uploaded into Ingenuity Pathway Analysis which was used to generate the top canonical pathways involving the differentially regulated genes. Real-time PCR Gene expression analysis of whole blood-derived mRNA with HGU 133 Plus 2.0 chips was previously validated by real-time PCR8. We used real-time PCR to.