No abstract

Background and Purpose-We previously showed that mild hypothermia protects against experimental stroke, even when cooling was delayed by 2 hours. Protection may be due in part to inhibiting inflammation. To clarify, we examined leukocyte infiltration, microglial activation, and adhesion molecule expression in models of stroke and pure brain inflammation. Methods-Rats underwent 2-hour middle cerebral artery occlusion (MCAO; nϭ36) or intravenous injection with 5 mg/kg lipopolysaccharide (LPS; nϭ22). Temperature was lowered to 33°C for 2 hours or kept at 37°C. In MCAO, cooling was applied intraischemically or on reperfusion (delayed). In the LPS model, cooling began after injection. One and 3 days later, brains were assessed for neutrophils, monocytes/microglia, major histocompatibility complex class II antigen, and intercellular adhesion molecule-1 (ICAM-1). Results-One

Prior studies had shown that the Mirk/dyrk1B gene is amplified/upregulated in about 75% of ovarian cancers, that protein levels of this kinase are elevated in quiescent G0 cells, and that Mirk maintains tumor cells in quiescence by initiating rapid degradation of cyclin D isoforms and by phosphorylation of a member of the DREAM complex. Depletion of Mirk/dyrk1B led to increased cyclin D levels, an elevated ROS content, and loss of viability. However, many normal cells in vivo are quiescent, so targeting a kinase found in quiescent cells might be problematic. In the current study, Mirk kinase activity was found to be higher in ovarian cancer cells than normal cells. Pharmacological inhibition of Mirk/dyrk1B kinase increased cyclin D levels both in quiescent normal diploid cells and in quiescent CDKN2A-negative ovarian cancer cells, but led to more active CDK4/cyclin D complexes in quiescent ovarian cancer cells, allowing them to escape G0/G1 quiescence, enter cycle with high ROS levels and undergo apoptosis. The ROS scavenger N-acetyl cysteine reduced both the amount of cleaved PARP and the extent of cancer cell loss. In contrast, normal cells were spared because of their expression of CDK inhibitors that blocked unregulated cycling. Quiescent early passage normal ovarian epithelial cells and two strains of quiescent normal diploid fibroblasts remained viable after inhibition of Mirk/dyrk1B kinase, and the few cells that left G0/G1 quiescence accumulated in G2+M. Thus inhibition of Mirk kinase targeted quiescent ovarian cancer cells.

P84 BACKGROUND: Inflammation potentiates ischemic injury especially with reperfusion, while mild hypothermia is an effective neuroprotectant. We studied whether mild hypothermia’s protective effect may be due to blunting the inflammatory response. METHODS: We compared endothelial intracellular adhesion molecule (ICAM-1) expression, neutrophil & monocyte infiltration, and microglia activation under normothermic (38C n=24) and hypothermic (intraischemic brain cooling to 33C for 2h n=24) conditions 1,3, and 7 days following transient (2h) focal cerebral ischemia in rats. ICAM-1, ED-1 (to detect cells of monocyte lineage including activated microglia) and myeloperoxidase (MPO, to identify neutrophils) were detected using immunohistochemistry on brain sections. We measured cell densities of ICAM-1, ED-1, and MPO in the peri-infarct region. Infarct size was also measured from histology derived sizes of the ipsilateral hemisphere. RESULTS: Mild hypothermia reduced infarct size 1–7 days after stroke onset. 1d: 38C: 28±12%, 33C 8±6%; 3d: 38C: 39±23%, 33C: 11±11%; 7d: 38C: 37.3±5.8% vs 33C: 19.4±5.7% (p<0.05). The number of ICAM-1 positive vessels per high power field (HPF) decreased under hypothermia 1–7 days later. 1d: 38C: 35.8±1.7 vs 33C: 24.4±1.4 (p<0.01); 3d: 38C: 36.6±5.0 vs 33C: 23.5 1.3 (p=0.07); 7d: 38C: 69.9±2.7, 33C: 43±5.2 (p<0.001). In addition, neutrophil density (cells/12 HPF) decreased under hypothermic conditions at 1 and 3 days. 1d: 38C: 48±3.0 vs 33C: 1.3±0.6 (p<0.001), 3d: 38C: 75±3.5 vs 33C: 20.3±8.1 (p<0.001). Monocyte and microglial density (cells/HPF) was decreased by mild hypothermia at 3 and 7, but not 1 day. 1d: 38C: 17.5±0.9, 33C: 16.6±0.8, NS; 3d: 38C: 45.5±1.7, 33C: 19.2±0.9, (p<0.0001); 7d: 38C: 75.0 ±2.6, 33C: 24.8±3.0, (p<0.0001). CONCLUSIONS: Mild hypothermia reduces adhesion molecule expression, acute (neutrophil) and subacute (monocyte) leukocyte infiltration and microglial activation. These changes are present even days after hypothermic treatment, and suggest that hypothermia significantly attenuates the inflammatory response.