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

工作总结：我们使用了31-P NMR 光谱学间接评估肌肉中血管生成 评估组织生物能量状态。我们发现， 测量结果对组织灌注的敏感性比 血压测量。此外，由于它们是 非侵入性，该方法也可用于人类受试者， 应用于实验动物的纵向研究。 虽然 肌肉的生物能状态不是直接测量 血流量，增加肌细胞能荷与 更大的肌肉耐力，显然代表了一个理想的 治疗终点此外，直接比较 证明了生物能量和 通过31-P NMR测定的状态和通过31-P NMR测定的血流量 通过微球测量。 我们建立了一个 能获得31-P谱的实验装置 在动物的电刺激肌肉中。我们的实验 方案由测量细胞内磷酸盐组成， 腓肠肌的麻醉大鼠在休息，在一个 强烈的肌肉刺激期，以及从 刺激. 最初的调查集中在假设， 新生血管化发生得更慢， 与年轻动物相比，老年动物。我们发现 运动时磷酸肌酸和收缩力下降 明显更明显，他们的恢复显着 老年大鼠比年轻大鼠慢。这有力地表明 随着年龄的增长血管生成能力的丧失。 探讨蛋白酶体抑制剂 血管生成的调节剂，我们已经采用了协议， 将血管内皮生长因子（VEGF）递送至 缺血肢体一组实验涉及应用 VEGF在股动脉切除术前三周。这是 然后评估细胞内的能量电荷， 腓肠肌的肌肉。再一次，所有31-P NMR测量结合了生理应力， 探测血管储备。我们发现VEGF的作用本质上是 立即和深刻地规范模式， 对肌肉刺激和恢复的生物能响应， 这表明， 灌注血管 我们正在把核磁共振 直接测量血流的光谱数据， 来自微球测量。基于MRI的协议 血流测量也正在开发中。新软骨 通过核磁共振成像和光谱学的研究， 研究了在中空纤维生物反应器中生长的新软骨组织 （HFBR）系统，其允许软骨细胞和软骨细胞之间的相互作用。 他们精心设计的三维矩阵 以非侵入性方式纵向地持续几周。我们有 用它来评估胚胎软骨的发育， 鸡软骨细胞以及人关节软骨。 我们 进行了组织学和核磁共振之间的详细关联 来自鸡软骨细胞的软骨显微图像， 将NMR可测量参数与软骨相关联 微观结构我们还成功地开发了详细的 NMR衍生的组织性质与结果之间的相关性 生物化学分析。最后，我们目前正在纳入EPR 成像和光谱学到这些研究中，调查 线粒体代谢和局部氧含量 发育中的组织

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 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.