NMR STUDIES OF PHYSIOLOGY AND BIOCHEMISTRY IN CELLS, ORGANS AND ANIMALS

工作总结：我们已经使用31-P NMR光谱通过评估组织生物能量状态来间接评估肌肉中的血管生成。我们已经发现，这些测量比血压测量对组织灌注更敏感。此外，由于它们是非侵入性的，这些方法可以用于人类受试者，以及应用于实验动物的纵向研究。虽然肌肉的生物能量状态不是血流量的直接测量，但增加的肌细胞能荷与更大的肌肉耐力相关，并且清楚地代表了期望的治疗终点。此外，直接比较已经证明了由31-P NMR确定的生物能量状态与由微球测量确定的血流之间的高度相关性。我们已经建立了一个实验装置，它允许在电刺激的动物肌肉中获得31-P光谱。我们的实验方案包括测量的细胞内磷酸盐在腓肠肌的麻醉大鼠在休息时，在一段时间内的强烈的肌肉刺激，并在恢复刺激。最初的研究集中在一个假设，即与年轻动物相比，老年动物的新血管形成发生得更慢，更不完全。我们发现，磷酸肌酸和收缩力的下降与运动明显更明显，其恢复显着较慢，在老年大鼠比年轻大鼠。这强烈表明随着年龄的增长血管生成潜力的丧失。为了研究血管生成调节剂的作用，我们采用了向缺血肢体递送血管内皮生长因子（VEGF）的方案。一组实验涉及在股动脉切除前三周应用VEGF。随后在数周的时间内评估腓肠肌中的细胞内能荷。再一次，所有的31-P核磁共振测量纳入生理应力，以探测血管储备。我们发现，血管内皮生长因子基本上立即和深刻的正常化模式的生物能量响应肌肉刺激和恢复，这表明灌注血管的发展速度显着增加。我们已经发现，NMR光谱数据与从微球测量导出的血流的直接（侵入性）测量很好地相关。在其他工作中，我们正在研究在中空纤维生物反应器（HFBR）系统中生长的新软骨组织，该系统允许以非侵入性方式对软骨细胞及其精心制作的三维基质进行数周的纵向研究。我们已经用它来评估从胚胎鸡软骨细胞以及从人关节软骨的新软骨的发展。我们已经进行了详细的组织学和核磁共振显微图像之间的相关性，从鸡软骨细胞的软骨，相关的核磁共振可测量的参数与软骨显微结构。我们还成功地开发了NMR衍生的组织特性和生化测定结果之间的详细相关性。最后，我们目前正在将EPR成像和光谱学纳入这些研究中，研究发育组织中的线粒体代谢和局部氧含量。- 肌肉、血管生成、软骨、骨关节炎

Summary 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 have found that the NMR spectroscopy data correlates well with direct (invasive) measurement of blood flow as derived from microsphere measurements. In additional work, we are investigating 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. - muscle, angiogenesis, cartilage, osteoarthritis