Model Systems

  1. Selective assay for S-nitrosoproteins with picomolar resolution: We developed a modification of a standard assay to permit use in presence of transition metals in biomaterials. This level of resolution and correction of metal artifact is necessary to explore NO-Hb chemistry under physiological conditions.
  2. Thin film tonometer for manipulating dissolved gases in aqueous solutions or cellular suspensions: This is a custom designed device designed precisely manipulate dissolved gases in solutions, plasma, or RBC suspensions so that we may: 1) characterize coupling between RBC nitrosothiol content and HbSO2, 2) demonstrate transfer of intraerythrocytic NO equivalents to extraerythrocytic thiols, and 3) map plasma transnitrosation cascades. The relative abundance of thiol redox couples are easily manipulated in this system and circulatory transit can be simulated by precisely manipulating dissolved gas concentrations over time. Most importantly, the solution may be sampled without exposure to ambient gas.
  3. Hypoxia Chamber: Commercially available temperature/humidity controlled “glove box” for handling samples in a controlled gas environment – permits handling & processing of samples without altering dissolved gases.
  4. Hollow Fiber cell culture system: Commercially available cell culture “bioreactor” system (FiberCell) in which endothelial cells are grown to line the inner lumen of 700 micron semi-permeable fibers; 20 fibers are clustered in a cartridge. The cartridge has access ports to the luminal and extra-luminal space (like a dialysis cartridge). Culture media is pumped through the luminal space simulating flow through the microcirculation; we have uniquely adapted this system by introducing countercurrent gas flow through the extraluminal space to create an O2 gradient – to simulate capillary traversal through living tissue by introducing RBC suspensions into the luminal flow of culture media. We are developing this “mock microcirculation” to study (under physiological conditions) transnitrosation reactions between desaturating RBCs and endothelial cell surface targets and subcellular compartments.
  5. Murine Pulmonary and Cardiovascular Physiologic Platform: This is a bioassay set to designed to quantify the physiologic impact of data generated in the model systems above. Centralized electronics, signal conditioning, computer, and software support the following experimental systems:
    1. Vascular Ring System: Standard commercially available system in which cut vascular rings are suspended between force transducers submerged in physiologic buffer. It is used for precisely monitoring response of vascular tone to pharmacologic manipulation. Gas tension in the buffer is easily controlled and permits easy quantification of O2 dependent RBC vasoactivity.
    2. Murine Cardiac Cath System: Standard system designed to measure RV or LV pressure in lightly anesthetized, spontaneously breathing mice.
    3. Murine Isolated Perfused Lung System: Customized commercially available system in which O2 gradients can be created and manipulated across an entire vascular bed whilst monitoring physiologic vascular response, gas exchange, acquiring system efflux for assay, and generating tissue for study. Adapted to manipulate 1) inhaled gas composition, 2) delivery of inhaled drugs, 3) continuous monitoring of dissolved oxygen in pulmonary venous efflux 4) manipulation of dissolved gases in PA perfusate, and 4) drug (or erythrocyte) delivery into PA perfusate stream.
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