of Work: We have used 31-P NMR spectroscopy to indirectly assess angiogenesis in muscle by evaluating tissue bioenergetic status. We have found that these measurements are much more sensitive to tissue perfusion than are blood pressure measurements. In addition, because they are non-invasive, the methods can be used for human subjects, as well as applied to longitudinal studies in experimental animals. Although the bioenergetic state of muscle is not a direct measurement of blood flow, increased myocellular energy charge correlates with greater muscular endurance, and clearly represents a desirable therapeutic endpoint. Further, direct comparisons have demonstrated a high degree of correlation between bioenergetic status, as determined by 31-P NMR, and blood flow as determined by microsphere measurements. We have constructed an experimental apparatus which permits 31-P spectra to be obtained in electrically stimulated muscle in animals. Our experimental protocol consists of measurement of intracellular phosphates in the gastrocnemius muscle of the anesthetized rat at rest, during a period of intense muscle stimulation, and during recovery from stimulation. Initial investigations focused on the hypothesis that neovascularization takes place more slowly and less completely in aged animals as compared to young animals. We found that the declines in phosphocreatine and in contraction force with exercise are significantly more pronounced, and their recovery significantly slower, in the older than in the younger rats. This strongly suggests the loss of angiogenic potential with age. To study the effect of modulators of angiogenesis, we have employed protocols for delivery of vascular endothelial growth factor (VEGF) to the ischemic limb. One set of experiments involved application of VEGF three weeks prior to femoral artery resection. This was followed by assessment of intracellular energy charge in the gastrocnemius muscle over a period of weeks. Once again, all 31-P NMR measurements incorporated physiologic stress in order to probe vascular reserve. We found that VEGF acted essentially immediately and profoundly to normalize the pattern of bioenergetic response to muscle stimulation and recovery, suggesting a marked increase in the rate of development of perfusing vessels. We are in the process of correlating our NMR spectroscopy data with direct measurement of blood flow as derived from microsphere measurements. Protocols for MRI-based blood flow measurements are also being developed. Neocartilage studies by NMR imaging and spectroscopy We have also investigated neocartilage tissue grown in a hollow fiber bioreactor (HFBR) system which permits chondrocytes and the three-dimensional matrix which they elaborate to be studied longitudinally for several weeks in a non-invasive manner. We have used this to assess the development of neocartilage from embryonic chick chondrocytes as well as from human articular cartilage. We have carried out detailed correlations between histology and NMR microimages in cartilage derived from chick chondrocytes, correlating NMR-measurable parameters with cartilage microstructure. We have also successfully developed detailed correlations between NMR-derived tissue properties and the results of biochemical assays. Finally, we are currently incorporating EPR imaging and spectroscopy into these studies, investigating mitochondrial metabolism and local oxygen content in the developing tissue.
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