HISTAMINE CONTAINING CENTRIFUGAL AXONS IN THE RETINA

这是一项探索性研究补助金(R21)的建议，是对RFA DK 98 009关于糖尿病并发症的发病机制和治疗的回应。这项拟议研究的长期目标是了解从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中所起的作用。在初步实验中，猕猴视网膜中来自视神经而不是细胞体的轴突被发现含有组胺，它们的形态以及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。这些结果提供了更多证据，证明在灵长类动物中确实存在离心轴突。大脑中唯一含有组胺的神经元在下丘脑后部被发现，逆行标记研究表明离心性轴突起源于下丘脑后部。结果还表明，组胺及其拮抗剂可以用来研究正常灵长类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标是开发一种用于电子显微镜的离心轴突标记方法，并研究它们的超微结构。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进行专门接触，这些将被描述。电子显微镜免疫组织化学的三个可能的标记是：神经肽甘丙素，它定位于灵长类动物中含有组胺的神经元和猕猴视网膜中的轴突，但不定位于细胞体；组氨酸脱羧酶，合成组胺的酶，以及组胺本身。这三种物质以前都通过电子显微镜定位过，但没有在视网膜中定位。第二个具体目标是确定人类离心性轴突是否也含有组胺，正如初步数据所预期的那样，并描述糖尿病捐赠者眼睛中这些轴突的任何变化。工作假设是，糖尿病患者的这些轴突比正常情况下更活跃，这就是为什么抗组胺药物在防止这些患者的血管渗漏方面有效。如果这是正确的，那么这种增加的活动可能存在形态上的关联。人类材料也将使用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.