Effects of moderate and deep hypothermia on RNA-binding proteins RBM3 and CIRP expressions in murine hippocampal brain slices.

Therapeutic hypothermia has emerged as an effective neuroprotective therapy for cardiac arrest survivors. There are a number of purported mechanisms for therapeutic hypothermia, but the exact mechanism still remains to be elucidated. Although hypothermia generally down-regulates protein synthesis and metabolism in mammalian cells, a small subset of homologous (>70%) cold-shock proteins (RNA-binding motif protein 3, RBM3 and cold-inducible RNA-binding protein, CIRP) are induced under these conditions. In addition, RBM3 up-regulation in neuronal cells has recently been implicated in hypothermia-induced neuroprotection. Therefore, we compared the effects of moderate (33.5°C) and deep (17°C) hypothermia with normothermia (37°C) on the regulation of RBM3 and CIRP expressions in murine organotypic hippocampal slice cultures (OHSC), hippocampal neuronal cells (HT-22), and microglia cells (BV-2). Moderate hypothermia resulted in significant up-regulation of both RBM3 and CIRP mRNA in murine OHSC, but deep hyporthermia did not. RBM3 protein regulation was also significantly up-regulated by 33.5°C, but no significant up-regulation of CIRP protein was observed in the OHSC. Additionally, OHSC exposed to 17°C for 24h were positive for Propidium Iodide (PI) immunostaining, indicating the onset of cell death. Similarly, RBM3 gene expression in a HT-22 neuronal cells mono-culture and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were also up-regulated at 33.5°C but only in the co-culture at 17°C. No significant up-regulation of RBM3 nor CIRP gene expression were observed in a BV-2 mono-culture at either temperature. We observed that RBM3 mRNA and protein expressions in murine OHSC, as well as in mono-culture of HT-22 neuronal cells and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were significantly up-regulated by exposure to moderate hypothermia. These findings further support the implication of RBM3 as a potential effector for hypothermia-induced neuroprotection.

An operational concept for long-term cinemicrography of cells in mono- and co-culture under highly controlled conditions--the SlideObserver.

Cell morphology, proliferation and motility, as well as mono- and heterotypic cell-to-cell interactions, are of increasing interest for in vitro experiments. However, tightly controlling culture conditions whilst simultaneously monitoring the same set of cells is complicated. Moreover, video-microscopy of distinct cells or areas of cells over a prolonged period of time represents a technical challenge. The SlideObserver was designed for cinemicrography of cells in co-and monoculture. The core elements of the system are the SlideReactors, miniaturised hollow fibre-based bioreactors operated in closed perfusion loops. Within the SlideReactors, cells can be cultured under adaptable conditions as well as in direct- and indirect co-culture. The independent perfusion loops enable controlled variation of parameters such as medium, pH, and oxygenation. A combined automated microscope stage and camera set-up allows for micrograph acquisition of multiple user-defined regions of interest within the bioreactor units. For proof of concept, primary cells (HUVEC, human hepatocytes) and cell lines (HuH7, THP-1) were cultured under stable and varying culture conditions, as well as in mono- and co-culture. The operational system enabled non-stop imaging and automated control of process parameters as well as elective manipulation of either reactor. As opposed to non-perfused culture systems or comparable devices for cinemicrographic analysis, the SlideObserver allows simultaneous morphological monitoring of an entire culture of cells in multiple bioreactors.

Establishment of a coculture model for studying inflammation after pediatric cardiopulmonary bypass: from bench to bedside.

Cardiopulmonary bypass (CPB) has been known to induce an inflammatory response that is influenced by various factors. Hypothermia is supposed to reduce inflammation after CPB. We developed an in vitro coculture model for CPB and compared the effects of hypothermia on the inflammatory response in the coculture model with results from a clinical prospective randomized trial. The coculture model consisted of endothelial cells and monocytes. Cells were stimulated with tumor necrosis factor (TNF)-α and exposed to deep hypothermia (20°C) or normothermia (37°C). In the clinical trial, 20 patients undergoing CPB for ventricular septum defect receive either normothermic (37°C) or mild hypothermic (32°C) CPB. We observed a significant interleukin (IL)-6 and IL-8 release in the coculture model 2 and 24 h after the experimental start. In the clinical trial, cytokines were significantly increased directly after weaning from CPB and remained elevated until 24 h. IL-8 and IL-6 secretions were similar in the hypothermic and normothermic group of the coculture model and the patients after 24 h. These results demonstrate that the inflammatory reaction observed in our coculture model is comparable with the cytokine increase in the blood of children undergoing CPB. Our coculture model could be useful for studies on the mechanisms of CPB-induced inflammation.

How does hypothermia protect cardiomyocytes during cardioplegic ischemia?

OBJECTIVE: Insufficient myocardial protection is still a considerable cause for in-hospital mortality in children. The purpose of our study was to investigate underlying the basic mechanisms of cardioplegic cardioprotection during hypothermic and normothermic ischemia in a cardiomyocyte cell culture model. METHODS: We cooled cardiomyocytes to 20°C for 20min; during this time, cardiac arrest was simulated by oxidative damage with 2mM H2O2 and cardioplegic solution, followed by rewarming to 37°C. Later on, we analyzed cardiomyocyte cell morphology (phase-contrast-microscopy), viability (trypan blue staining), inflammation (cyclooxygenase-2 (Cox-2) and phosphorylated-extracellular signal-regulated kinase (pERK) 1/2 expression in Western blot analysis), and expression of Akt survival protein (Western blot technique). RESULTS: Hypothermia increases cell survival of cardiomyocytes after cardioplegic ischemia, as demonstrated in significantly higher cell viability and less cell death in these cells compared with normothermic H2O2-damaged cardiomyocytes. As a possible underlying cellular mechanism, we found that, during cold cardioplegic ischemia, ERK 1/2 enzyme is less phosphorylated than under conditions of normothermic cardioplegic ischemia. This is in line with significantly diminished Cox-2 expression during cold cardioplegic ischemia. Moreover, hypothermic cardioplegia preserved cell survival by upregulation of Akt transcription factor in cardiomyocytes. CONCLUSION: In the present cell culture study, we clearly demonstrated that hypothermia exerts additional protection for cardiomyocytes during cardioplegic ischemia. The understanding of underlying basic mechanisms is evident to improve current techniques of myocardial protection.