Acute ischemia followed by reperfusion can lead to both interstitial and intracellular edema in skeletal muscle of the extremities. If severe, the edema can lead to compressive decreases in tissue perfusion and loss of the limb. The mechanism by which the initial transient edema associated with ischemia and reperfusion develops into a severe and sustained compressive edema is not known. This proposal is designed to test the hypothesis that neutrophil-mediated degradation of hyaluronan in skeletal muscle leads to diminished convective transport of albumin within the interstitium and a prolonged extravascular washout of albumin. The disruption of aggregated hyaluronan reduces hydrated pathways for protein movement within the interstitium even though there is increased matrix hydration due to the edema. The in vivo model used to evaluate this hypothesis will be anesthetized rabbit following tourniquet ischemia in which prenodal lymph draining the skeletal muscles of the lower leg can be collected. Hyaluronan degradation will be determined from measurements of the change in hyaluronan flux and size of the lymph since lymph drainage is a major component in hyaluronan metabolism. Additional measurements will be obtained on the change in hyaluronan size in skeletal muscle samples taken from the lower leg. Measurements of tissue myeloperoxidase activity will be used to determine the presence of neutrophils. The temporal relationship between the length of ischemia, the cellular release of myoglobin, and the degree of hyaluronan degradation will be carefully evaluated, since myoglobin may provide a major source of the extracellular iron required for hyaluronan degradation. The degree of interstitial edema, estimated from change in the extravascular distribution space for Cr-EDTA and mass for native albumin, will be correlated to the changes in hyaluronan size. The influence of ischemia-reperfusion on the time dependent, extravascular washout of albumin will be studied during increased venous pressure by subtracting the clearance for extravascular uptake from the lymph clearance. The influence of hyaluronan degradation on interstitial and lymph albumin transport will be studied using local intra- arterial infusion of testicular hyaluronidase. Measurements of the extravascular washout of albumin following hyaluronidase pretreatment will be compared to those following ischemia-reperfusion. The influence of hyaluronidase pretreatment on interstitial convective transport for albumin will be studied by comparing the rate of labeled albumin equilibration between plasma, tissue and lymph. Under in vitro conditions, myoglobin will be compared to free iron in its ability to catalyze hyaluronan degradation by activated polymorphonuclear leukocytes. Additionally, the combined influence of hyaluronan and collagen on superoxide production by activated neutrophils will be measured in order to determine if the neutrophil response in the interstitium is amplified by the presence of these substances in the extracellular matrix. Additional in vivo and in vitro studies will determine if a lipid soluble anti-oxidant is more effective than a small, water soluble anti-oxidant or iron chelator in preventing hyaluronan degradation and enhancing the time dependent, extravascular washout of albumin following ischemia-reperfusion. Such experiments will provide insight as to the potential beneficial effects of these agents following limb ischemia and reperfusion.
| Armstrong, Shayn E; Bell, Donald R (2002) Relationship between lymph and tissue hyaluronan in skin and skeletal muscle. Am J Physiol Heart Circ Physiol 283:H2485-94 |
| Armstrong, Shayn E; Bell, Donald R (2002) Measurement of high-molecular-weight hyaluronan in solid tissue using agarose gel electrophoresis. Anal Biochem 308:255-64 |
| Armstrong, Shayn E; Bell, Donald R (2002) Ischemia-reperfusion does not cause significant hyaluronan depolymerization in skeletal muscle. Microvasc Res 64:353-62 |
| Kupinski, A M; Bock, D E; Bell, D R (1997) Skeletal muscle ischemia-reperfusion causes transitory increase in microvascular protein permeability. Am J Physiol 273:H303-9 |
