The local and systemic consequences of limb ischemia form the general framework of this laboratory's long term investigative interests. The current goal is to test the thesis that thromboxane (Tx) A2 synthesized after ischemia will moderate many post-ischemic changes. Possible direct TxA2 effects are due to its vasoactive and putative vasotoxic actions. Indirect effects may relate to the proaggregatory activity of TxA2 on platelets and white blood cells (WBC), causing sequellae due to the local and pulmonary entrapment of these cells. The first section of this proposal will quantitate the role of circulating and parenchymal cells in Tx synthesis in the dog hind limb subjected to ischemia, as well as in in vitro cells grown in culture subjected to hypoxia and acidosis. Exhaustion of the synthetic capacity of TxA2 and its antagonist prostacyclin (PGI2), following prolonged and repeated ischemic stimuli, will be monitored by radioimmunoassay of plasma concentrations of the hydrolysis products of TxA2 and PGI2. The longest ischemic interval tested will be 2 h, a period which does not lead to permanent injury. That TxA2 and PGI2 may influence both the duration of post-ischemic hyperemic flow, as well as flow distribution to skin, muscle and arterio-venous shunts will be studied using microsphere injections in ischemic hind limbs of animals treated with inhibitors of TxA2 and PGI2 synthesis. Since the proaggregating properties of TxA2 may lead to indirect effects, platelets labeled with 111-Indium will be used to test whether ischemia leads to the local or pulmonary sequestration of these cells. In order to document whether the pulmonary vascular response to ischemia relates to entrapped platelet aggregates, ischemia studies will be conducted in platelet depleted dogs. It is thought that TxA2 moderates microvascular permeability. This will be examined in dogs in whom a popliteal lymphatic channel has been cannulated. Changes in lymph flow and lymph/plasma protein ratios following ischemia will be contrasted to changes induced by increased venous pressure. Whether or not increased permeability is due to increased TxA2, reduced PGI2 or is indirectly related to WBC entrapment will be studied in animals treated with Tx and cyclooxygenase inhibitors and those depleted of WBC. Additional studies will be done where evidence of acridine orange labeled WBC entrapment and enzyme release in the ischemic hind limb will be sought. Taken as a whole, these experiments are designed to assay the direct and indirect roles of TxA2 synthesized in response to ischemia.
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