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

工作概述：我们已经使用31-P核磁共振波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，也可以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，但增加的心肌细胞能量电荷与更大的肌肉耐力相关，显然代表了理想的治疗终点。此外，直接比较表明，生物能量状态（由31-P核磁共振测定）与血流量（由微球测量测定）之间存在高度相关性。我们已经建造了一个实验装置，可以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测量麻醉大鼠腓肠肌细胞内磷酸盐在休息时，在一段时间的强烈肌肉刺激，并从刺激中恢复。最初的调查集中在一个假设上，即与幼龄动物相比，老年动物的新生血管形成速度更慢，更不完全。我们发现，与年轻的大鼠相比，年老的大鼠的磷酸肌酸和收缩力的下降更为明显，恢复速度也要慢得多。这强烈表明血管生成潜力随着年龄的增长而丧失。为了研究血管生成调节剂的作用，我们采用了将血管内皮生长因子（VEGF）输送到缺血肢体的方案。一组实验涉及在股动脉切除术前三周应用VEGF。随后在数周内评估腓肠肌细胞内能量电荷。再一次，所有的31-P核磁共振测量都包含了生理压力，以探测血管储备。我们发现血管内皮生长因子对肌肉刺激和恢复的生物能量反应模式起着立即而深刻的作用，这表明灌注血管的发育速度显着增加。我们已经发现，核磁共振波谱数据与直接（侵入性）测量血液流量的微球测量有很好的相关性。在额外的工作中，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，该系统允许以无创方式纵向研究软骨细胞和它们精心制作的三维基质数周。我们已经用这种方法来评估从鸡胚胎软骨细胞和人关节软骨中形成新软骨的情况。我们在鸡软骨细胞衍生的软骨中进行了组织学和核磁共振显微图像之间的详细相关性，将核磁共振可测量参数与软骨微观结构相关联。我们还成功地开发了核磁共振衍生组织特性与生化分析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，研究线粒体代谢和发育组织中的局部氧含量。-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