High-performance liquid chromatography (HPLC) quantification of catecholamines

The spleen was collected 1, 6, and 24 h after SCI, the tissue was divided in two, one half was used for HPLC, and another half was used for real-time polymerase chain reaction (qPCR), both samples were rapidly snap-frozen in liquid nitrogen and stored at − 80 °C. The spleens were weighed, and then, catecholamines were extracted after homogenization in 200 μL of perchloric acid. Samples were sonicated and then centrifuged at 10,000 rotations per minute (RPM) for 10 min. Resulting supernatants were filtered through a Spin-X HPLC column (Costar) to remove debris. Levels of dopamine, NE, epinephrine, and serotonin were measured by HPLC combined with electrochemical detection using a Gilson instrument, fitted with an analytical column (Supelco Supelcosil LC-18 3 mM, flow rate: 10 ml/min) as previously described [20]. Briefly, 150 ml supernatant aliquots were injected into the system, using a mobile phase of 0.7 M aqueous potassium phosphate (pH 3.0) in 10% methanol, 1-heptanesulfonic acid (222 mg/l), and Na-EDTA (40 mg/l). A standard curve using known concentrations of NE, epinephrine, and dopamine was run.