Previous studies have demonstrated a role for both tumor necrosis factor (TNF) and reactive oxygen intermediates (ROI) in hepatic ischemia/reperfusion (I/R) injury. Biologically active TNF was present in liver homogenates in ischemic and nonischemic lobes after 2 h of ischemia but without reperfusion. Using an in situ liver perfusion model, we measured ROI, TNF, and hepatic enzymes in the effluent after 2 h of ischemia. Increased reduction of ferricytochrome C was observed in the hepatic effluent, indicative of the formation of ROI. Treatment of animals with TNF neutralizing antisera significantly reduced both ROI and aspartate aminotransferase (AST). Animals treated with superoxide dismutase (SOD), or SOD + catalase (CAT) had greater TNF in the hepatic effluent compared with I/R alone; however, SOD or SOD + CAT did not cause additional release of AST.SOD + CAT plus anti-TNF serum resulted in significant protection compared with SOD + CAT plus control serum. Reperfusion of ischemic liver with 4 mM H2O2 increased both TNF and AST. Optimal protection of hepatocellular injury from reperfusion injury is achieved with a combination of antioxidants and inhibition of TNF.
Because many types of living cells are sensitive to applied strain, different in vitro models have been designed to elucidate the cellular and subcellular processes that respond to mechanical deformation at both the cell and tissue level. Our focus was to improve upon an already established strain system to make it capable of independently monitoring the deflection and applied pressure delivered to specific wells of a commercially available, deformable multiwell culture plate. To accomplish this, we devised a custom frame that was capable of mounting deformable 6 or 24 well plates, a pressurization system that could load wells within the plates, and a camera-based imaging system which was capable of capturing strain responses at a sufficiently high frame rate. The system used a user defined program constructed in Labview R to trigger plate pressurization while simultaneously allowing the deflection of the silicone elastomeric plate bottoms to be imaged in near real time. With this system, up to six wells could be pulsed simultaneously using compressed air or nitrogen. Digital image capture allowed near-real time monitoring of applied strain, strain rate, and the cell loading profiles. Although our ultimate goal is to determine how different strain rates applied to neurons modulates their intrinsic biochemical cascades, the same platform technology could be readily applied to other systems. Combining commercially available, deformable multiwell plates with a simple instrument having the monitoring capabilities described here should permit near real time calculations of stretchinduced membrane strain in multiple wells in real time for a wide variety of applications, including high throughput drug screening.
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