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

细胞、器官和动物生理学和生物化学的核磁共振研究

• 批准号: 6431420
• 负责人: RICHARD SPENCER
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431420
• 关键词: age difference; biochemistry; cartilage; cartilage metabolism; chemical kinetics; clinical research; computer simulation; exercise; hormone regulation /control mechanism; human subject; method development; muscle; muscle metabolism; nuclear magnetic resonance spectroscopy; physiology

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.

工作摘要：我们使用31-P NMR光谱法通过评估组织生物能状态来间接评估肌肉中的血管生成。 我们发现，与血压测量相比，这些测量对组织灌注更为敏感。 另外，由于它们是非侵入性的，因此这些方法可用于人类受试者，并用于实验动物的纵向研究。 尽管肌肉的生物能状态不是血流的直接测量，但增加的心肌电荷与更大的肌肉耐力相关，并且显然代表了理想的治疗终点。此外，直接比较表明，由31-P NMR确定的生物能状态与通过微球测量确定的血流之间的相关性很高。我们构建了一个实验设备，该设备允许在动物的电刺激肌肉中获得31-P光谱。 我们的实验方案包括在静止大鼠的胃内肌肉中测量细胞内磷酸盐，在强烈的肌肉刺激期间以及从刺激中恢复期间。初步研究的重点是与年轻动物相比，在老年动物中，新生血管化的发生较慢，更少。我们发现，与年轻大鼠相比，磷酸甲酸和运动收缩力的下降明显更为明显，并且它们的恢复速度明显慢。 这强烈表明随着年龄的增长而丧失血管生成潜力。为了研究血管生成调节剂的作用，我们采用了方案将血管内皮生长因子（VEGF）传递到缺血性肢体。 一组实验涉及在股动脉切除前三周使用VEGF。 随后在几周内评估了胃肌肉肌的细胞内能电荷。 再次，所有31-P NMR测量都融合了生理压力，以探测血管储备。 我们发现，VEGF基本上立即采取了行动，以使对肌肉刺激和恢复的生物能反应的模式正常化，这表明灌注血管的发育速度显着增加。 我们发现，NMR光谱数据与从微球测量得出的血流直接（侵入性）测量很好地相关。 在其他工作中，我们正在研究在空心纤维生物反应器（HFBR）系统中生长的新能力组织，该组织允许软骨细胞和三维基质，它们会以无侵入性的方式进行纵向研究几周。 我们已经用它来评估胚胎鸡软骨细胞以及人类关节软骨的新能力的发展。 我们已经在源自雏鸡软骨细胞的软骨中进行了组织学与NMR微图像之间的详细相关性，将NMR可测量参数与软骨微结构相关联。 我们还成功地开发了NMR衍生的组织特性与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，研究了发育中的组织中的线粒体代谢和局部氧含量。