Neural-Vascular Interactions in the Retina

描述（由申请人提供）：本研究计划的主要目标是增加对控制系统的理解，该控制系统将视网膜代谢负荷与视网膜循环的营养供应联系起来。在这里提出的实验中，两种不同的方法测量视网膜血流量，荧光微球法和粒子跟踪法，将被用来在正常条件下，在视网膜的光刺激期间，在大鼠的血流量测量。本研究的具体目的是1）用于测量通过大鼠视网膜循环的血流量的粒子跟踪方法的改进和表征，2）用于测量通过大鼠视网膜循环的血流量的荧光微球方法的改进和表征，3）视网膜的闪烁刺激对视网膜血流量的影响的表征，和4）确定视网膜内动作电位产生在视网膜刺激期间触发增加的血流量中所起的作用。 用于血流测量的微球方法是将标记的微球注射到麻醉大鼠的左心室中并嵌入视网膜中的技术。处死动物后，取出视网膜并进行整体计数，使用荧光显微镜对球体进行计数。在粒子追踪技术中，静脉内注射非常小的微球（直径约2 μ m）。使用带有荧光附件的显微镜对这些颗粒进行成像。将图像记录在sVHS录像带上，并计算血液速度和流量。 一旦这些测量技术的可变性和再现性被优化用于大鼠视网膜，将研究神经活动对视网膜血流的影响。首先，将在大鼠视网膜中表征伴随闪烁刺激的视网膜血流量的增加。在随后的实验中，Na+/K+泵阻断剂TTX将用于阻断视网膜内层神经元中的动作电位产生，以观察这种活动是否会触发视网膜血流的增加，这种增加是对视网膜刺激的反应。

DESCRIPTION (provided by applicant): This research program's major goal is to increase understanding of the control system that links retinal metabolic load with nutrient supply from the retinal circulation. In the experiments proposed here, two different methods of measurement of retinal blood flow, the fluorescent microsphere method and the particle tracking method, will be used to make blood flow measurements in rats during normal conditions and during photic stimulation of the retina. The specific aims of this study are 1) Modification and characterization of the particle tracking method for measurement of blood flow through the retinal circulation of the rat, 2) Modification and characterization of the fluorescent microsphere method for the measurement of blood flow through the retinal circulation of the rat, 3) Characterization of the effect of flicker stimulation of the retina on retinal blood flow, and 4) Determination of the role that action potential generation in the inner retina plays in triggering increased blood flow during retinal stimulation. The microsphere method for blood flow measurement is a technique in which labeled microspheres are injected into the left ventricle of an anesthetized rat and become embedded in the retina. After the animals are sacrificed, the retina is removed and wholemounted, and the spheres are counted using a fluorescent microscope. In the particle tracking technique, very small microspheres (roughly 2 um in diameter) are injected intravenously. These particles are imaged using a microscope with a fluorescence attachment. Images are recorded on sVHS videotape and blood velocities and flows are calculated. Once the variability and reproducibility of these measurement techniques are optimized for use in the rat retina, the effects of neural activity on retinal blood flow will be investigated. First of all, the increase in retinal blood flow that accompanies flicker stimulation will be characterized in the rat retina. In later experiments, the Na+/K+ pump blocker TTX will be used to block action potential generation in inner retinal neurons to see if this activity triggers the increase in retinal blood flow that occurs in response to retinal stimulation.