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

工作总结：我们已经使用了31-P核磁共振 光谱学间接评估肌肉血管生成的方法 评估组织的生物能量状态。我们发现这些 测量结果对组织灌注量的敏感性比 测量血压。此外，因为他们是 这种方法是非侵入性的，也可以用于人类受试者 应用于实验动物的纵向研究。虽然 肌肉的生物能量状态不是对 血流，心肌细胞能量电荷增加与 更大的肌肉耐力，显然代表着一种理想的 治疗终点。此外，直接比较已经 证明了生物能量学的高度相关性 由31-P核磁共振确定的状态和确定的血流量 通过微球测量。我们已经构建了一个 可获得~(31)P谱的实验装置 在电刺激的动物肌肉中。我们的实验 方案包括测量细胞内的磷酸盐 麻醉大鼠在静息状态下的腓肠肌 肌肉强烈刺激期，以及在恢复期间 刺激。最初的调查集中在这样的假设上 新生血管发生得更慢，也更不完全 与年轻动物相比，年老的动物。我们发现， 运动引起的磷酸肌酸和收缩力下降 明显更加明显，而且它们的恢复显著 年龄较大的大鼠比年轻的大鼠速度慢。这强烈地表明 血管生成能力随年龄增长而丧失。研究……的影响 血管生成的调节剂，我们已经采用了方案 血管内皮细胞生长因子(VEGFR)载体的构建 肢体缺血。其中一组实验涉及应用 血管内皮细胞生长因子在股动脉切除前三周。这是 然后是对细胞内能量电荷的评估 持续数周的腓肠肌。再一次，全是31-P 核磁共振测量包括生理应力，以便 探查血管储备。我们发现血管内皮细胞生长因子在本质上起作用 立即并深刻地实现 对肌肉刺激和恢复的生物能量反应， 表明……的发展速度显著提高。 灌流血管。我们正在将我们的核磁共振 直接测量血流量的光谱数据 由微球测量得出。基于磁共振成像的协议 血流量测量也在开发中。新地段 核磁共振成像和光谱学的研究我们还 研究了在中空纤维生物反应器中生长的新生组织。 (HFBR)系统，允许软骨细胞和 他们精心设计的待研究的三维矩阵 以一种非侵入性的方式纵向地持续数周。我们有 用它来评估从胚胎到新胚胎的发育。 鸡软骨细胞和人关节软骨细胞。我们 在组织学和核磁共振之间进行了详细的关联 来自小鸡软骨细胞的软骨显微图像， 核磁共振可测量参数与软骨的相关性 微观结构。我们还成功地开发了详细的 核磁共振组织特性与结果的相关性 生化分析的结果。最后，我们目前正在纳入EPR 对这些研究进行成像和光谱分析，调查 脑组织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.