HISTAMINE CONTAINING CENTRIFUGAL AXONS IN THE RETINA

这是一项探索性研究资助（R21）的申请，是为了响应RFA DK 98 009关于糖尿病并发症的发病机制和治疗的申请。该研究的长期目标是了解离心轴突释放的组胺在糖尿病患者视网膜小血管损伤的病因学中的作用。在初步实验中，猕猴视网膜上的视神经轴突，而不是细胞体，被发现含有组胺，它们的形态和与视网膜血管的接触，比其他选择性较低的染色方法更完整地描述出来。这些结果为离心轴突在灵长类动物中确实存在提供了额外的证据。大脑中唯一含有组胺的神经元位于下丘脑后部，逆行标记研究表明离心轴突起源于那里。研究结果还表明，组胺及其拮抗剂可用于研究正常灵长类动物视网膜中离心轴突的功能，并为已经观察到的组胺效应提供了解释。最后，这些发现可能具有临床意义，因为组胺拮抗剂显然可以防止糖尿病患者血视网膜屏障的破坏。提出的实验的第一个具体目的是开发一种方法来标记离心轴突的电子显微镜和研究他们的超微结构。猕猴视网膜中离心轴突的确切数量和直径将被确定，如果它们与视网膜血管或视网膜神经元有专门的接触，这些将被描述。电镜免疫组织化学的三个可能的标记是：神经肽甘丙氨酸，它已经定位于灵长类动物的含组胺的神经元和轴突，而不是猕猴视网膜的细胞体；组氨酸脱羧酶，合成组胺和组胺本身的酶。这三个病灶之前都通过电子显微镜定位过，但没有定位在视网膜上。第二个具体目标是确定人类离心轴突是否也含有组胺，正如从初步数据中预期的那样，并描述糖尿病供体眼睛中这些轴突的任何变化。工作的假设是，这些轴突在糖尿病患者中比正常人更活跃，这就是为什么抗组胺药能有效地防止这些患者的血管渗漏。如果这是正确的，那么这种活动的增加可能与形态学有关。人体材料也将使用在第一组实验中开发的电子显微镜技术进行研究。

This is a proposal for an Exploratory Research Grant (R21), submitted in response to RFA DK 98 009 on the Pathogenesis and Therapy of Complications of Diabetes. The long-term goal of the proposed research is to understand the role of histamine released from centrifugal axons in the etiology of small vessel damage to the retina of diabetic patients. In preliminary experiments, axons from the optic nerve, but not cell bodies, in the macaque retina were found to contain histamine, and their morphology and contacts with retinal blood vessels were described more completely than was possible with other, less-selective staining methods. These results provide additional evidence that centrifugal axons actually exist in primates. The only neurons in the brain that contain histamine are found in the posterior hypothalamus, where retrograde labeling studies indicate that the centrifugal axons originate. The results also suggest that histamine and its antagonists could be used to study the functions of the centrifugal axons in the normal primate retina, and they suggest explanations for the histamine effects that have been observed already. Finally, these findings may be clinically-significant since histamine antagonists apparently prevent the breakdown in the blood-retina barrier in diabetic patients. The first specific aim of the proposed experiments is to develop a method for labeling the centrifugal axons for electron microscopy and to study their ultrastructure. The exact number and diameters of the centrifugal axons in macaque retinas will be determined, and if they make specialized contacts with retinal blood vessels or retinal neurons, these will be described. Three possible marker for electron microscopic immunohistochemistry are: the neuropeptide galanin, which has been localized to histamine-containing neurons in primates and to axons, but not cell bodies, in the macaque retina; histidine decarboxylase, the enzyme that synthesizes histamine, and histamine, itself. All three have been localized previously by electron microscopy, but not in the retina. The second specific aim is to determine whether human centrifugal axons also contain histamine, as expected from the preliminary data, and to describe any changes in these axons in eyes of diabetic donors. The working hypothesis is that these axons are more active than normal in diabetics and that this is why antihistamines are effective in preventing vascular leakage in these patients. If this is correct, there may be a morphological correlate of this increased activity. The human material will also be studied using the electron microscopic technique developed in the first set of experiments.