Neural-Vascular Interactions in the Retina

描述(由申请人提供)：该研究项目的主要目标是增加对控制系统的了解，该系统将视网膜代谢负荷与来自视网膜循环的营养供应联系起来。在这里提出的实验中，将使用两种不同的测量视网膜血流量的方法--荧光微球法和颗粒跟踪法，来测量正常条件下和光刺激视网膜期间的大鼠的血流量。本研究的具体目的是：1)改良和表征测量大鼠视网膜循环血流量的颗粒追踪法；2)改良和鉴定测量大鼠视网膜循环血流量的荧光微球法；3)表征视网膜闪烁刺激对视网膜血流量的影响；4)确定视网膜内动作电位产生在刺激视网膜时触发血流量增加所起的作用。 血流测量的微球法是一种将标记的微球注入麻醉大鼠的左心室并嵌入视网膜的技术。动物被处死后，取出视网膜，用荧光显微镜对球体进行计数。在粒子示踪技术中，非常小的微球(直径约2微米)被静脉注射。这些颗粒使用带有荧光附件的显微镜进行成像。图像被记录在sVHS录像带上，并计算出血流速度和流量。 一旦这些测量技术的可变性和重复性被优化用于大鼠视网膜，就会研究神经活动对视网膜血流量的影响。首先，闪光刺激所伴随的视网膜血流量的增加将是大鼠视网膜的特征。在后来的实验中，Na/K泵阻滞剂TTX将被用来阻断视网膜内部神经元的动作电位产生，以了解这种活动是否会触发视网膜血流量的增加，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.