Mcp 1

Preoperative whole blood samples had previously been collected for each patient as a part of the Stephenson Cancer Center Biorepository (IRB#2555) and were utilized for this study. Following collection, the specimens underwent centrifugation and were stored in the University of Oklahoma biorepository. Enzyme-linked immunosorbent assay (ELISA) kits with internal controls were used to examine 25-OH Vitamin D (BD Biosciences, Beverly, MA), prealbumin (Abcam, Cambridge, MA), intracellular adhesion molecule 1 (ICAM-1), interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), monocyte chemoattractant protein 2 (MCP-2), macrophage derived chemokine (MDC), and tumor necrosis factor α (TNFα) (Bio-Rad Laboratories, Munich, Germany). All samples were run in duplicate and mean values were derived from a standard curve and reported.

At 24 h and 7 days after CCI or sham surgery, mice were anesthetized with urethane, the carotid artery exposed, severed, and blood collected from it, and the hemibrain ipsilateral to CCI/sham surgery was obtained. The arterial blood was centrifuged at 4500g for 10 min at 4 C and cytokine levels measured on the resulting serum. The hemibrain ipsilateral to CCI/sham surgery was homogenized in 1 ml of extraction buffer (0.01 M phosphate buffered saline (PBS; 2.7 mM potassium chloride, 0.137 M sodium chloride, pH 7.4) 2.7 mM, 1 mM EDTA, 1 mM PMSF, Protease Inhibitor cocktail) using a mini beadbeater set at 4800 RPM for 30 s to produce the PBS homogenate. A volume of 0.625 ml of the PBS homogenate was added to 0.125 ml of extraction buffer containing 0.6% Triton X-100 and centrifuged at 20,000g for 10 min at 4 C. Levels of brain cytokines were measured on the resulting supernatant. The multiplex kit for murine cytokines from BioRad was used to measure 23 cytokines: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12(p70), IL-13, IL-17, eotaxin (CCL11), G-CSF, GM-CSF, IFN-γ, KC (CXCL1), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), and TNF-α. Brain levels are reported relative to the protein content of the brain sample.

Total RNA was isolated and purified from each treatment group using RLT buffer (Qiagen) and the RNeasy mini kit (Qiagen, Valencia, CA). We then synthesized the complementary DNA using the iScript RT kit (Biorad). For cDNA from the ATII primary alveolar epithelial cell, we used custom QPCR plates (Biorad) to perform an analysis of IL-6st, MCP-1 (CCL2), and MIP-1β (CCL4). Additional primers for ER stress-related genes, CHOP and ATF4, were purchased from Integrated DNA Technologies. QPCR was performed using Sybr Green (Applied Biosystems) and the CFX96 Touch^™ Real-Time PCR Detection System (Biorad). Data were analyzed using the 2^−ΔΔCT method, and target genes were normalized to two housekeeping genes using ribosomal 18s and GAPDH.