maitotoxin and Disease-Models--Animal

Although hypothermia suppresses myocardial ischemia/reperfusion injury, whether it also protects the myocardium against cellular stresses such as chemical anoxia and calcium overload remains unknown. We examined the effect of mild hypothermia (33°C) on myocardial injury during ischemia/reperfusion, local administration of sodium cyanide (chemical anoxia), or local administration of maitotoxin (forced Ca overload) using cardiac microdialysis applied to the feline left ventricle. Baseline myoglobin levels (in ng/mL) were 237 ± 57 and 150 ± 46 under normothermia and hypothermia, respectively (mean ± SE, n = 6 probes each). Coronary artery occlusion increased the myoglobin level to 2600 ± 424 under normothermia, which was suppressed to 1160 ± 149 under hypothermia (P < 0.05). Reperfusion further increased the myoglobin level to 6790 ± 1550 under normothermia, which was also suppressed to 2060 ± 343 under hypothermia (P < 0.05). By contrast, hypothermia did not affect the cyanide-induced myoglobin release (930 ± 130 vs. 912 ± 62, n = 6 probes each) or the maitotoxin-induced myoglobin release (2070 ± 511 vs. 2110 ± 567, n = 6 probes each). In conclusion, mild hypothermia does not make the myocardium resistant to cellular stresses such as chemical anoxia and forced Ca overload.

Topics: Adenosine Triphosphate; Animals; Calcium; Cats; Cell Hypoxia; Disease Models, Animal; Heart Diseases; Hypothermia, Induced; Marine Toxins; Myocardial Reperfusion Injury; Myocardium; Myoglobin; Oxocins; Sodium Cyanide

MS-based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal-glial culture were subjected to excitotoxin N-methyl-D-aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH-L1, were first detected and assessed in the culture media by Western blot. The cell-conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data-dependent scanning but not UCH-L1. As a proof-of-principle study, rat glia-enriched cell cultures treated with MTX were used to investigate the time-dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial-enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS.

Topics: Animals; Apoptosis; Biomarkers; Brain Injuries; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Glial Fibrillary Acidic Protein; Marine Toxins; Mass Spectrometry; N-Methylaspartate; Necrosis; Neuroglia; Neurons; Oxocins; Protein Interaction Maps; Proteomics; Rats; Rats, Sprague-Dawley; Signal Transduction; Staurosporine; Ubiquitin Thiolesterase