The role of distal aqueous humor outflow tissue in glucocorticoid-induced glaucoma

项目总结/摘要 糖皮质激素常用于治疗炎症和自身免疫性疾病。虽然非常有效， 糖皮质激素可能在某些患者中诱发高眼压（OHT）/青光眼（他们被称为反应者; 其他称为无反应者）。小梁网（TM） 流出路径包含几种组织。从房水流出的上游到下游， 组织可分为近端流出组织，包括TM和Schlemm管，以及远端流出组织， 流出组织包括收集通道和水静脉。远端血流组织的重要性并不在于 在糖皮质激素诱导的OHT/青光眼中明确。因此，我们将研究我们的假设， 组织如TM也对糖皮质激素有不同的反应，这种反应导致OHT。我们 目的是阐明糖皮质激素诱导OHT的分子机制。我们提出两个具体目标。 SA 1.确定远端流出道组织是否差异调节GIG中的流出道阻力。使用我们的新 在灌注培养模型中，我们将在两个研究组中灌注成对的人供体角膜缘。A组：两者 轮辋将有TM使用Kahook双刀片删除。将用0.1%乙醇灌注一个边缘 （EtOH）作为媒介物对照，并且用100 nM地塞米松（DEX）对侧缘进行7-10天。IOP将为 记录并比较两眼之间的差异。将研究至少50对角膜缘。这个群体习惯于 确定远端流出组织是否能够引起糖皮质激素诱导的OHT。组B：一个轮辋将 直接灌注，而另一个边缘将去除TM组织，然后灌注。两个轮辋将 用DEX治疗7-10天，并记录IOP。将研究至少50对角膜缘。这 组用于确定远端流出组织是否与近端组织在糖皮质激素中同样重要， 诱导OHT。SA 2.比较近端和远端流出道组织之间的空间基因表达， 演出使用或不使用TM的成对角膜边缘将在一个边缘中用EtOH灌注，在另一个边缘中用DEX灌注。 这两个轮辋将用于空间基因表达研究。将固定组织并使用苏木精染色 和曙红来识别流出结构/区域。识别的区域将固定在载玻片上的芯片上。的 芯片含有5000个条形码点，这些点含有用于捕获、扩增和鉴定的寡核苷酸 用于RNA测序。芯片的分辨率足以研究近端和远端 流出组织将研究和比较应答者和非应答者组织。结果将 使用原位杂交和免疫染色进行验证。我们的项目是新颖的，因为我们将使用新的 角膜缘灌注培养模型和空间基因表达技术。这项研究意义重大，因为 它将为治疗糖皮质激素诱导的青光眼提供一种新的治疗靶点-远端流出组织。

PROJECT SUMMARY/ABSTRACT Glucocorticoids are frequently used to treat inflammatory and autoimmune diseases. Although very effective, glucocorticoids may induce ocular hypertension (OHT)/glaucoma in some patients (they are called responders; the others are called non-responders) after prolonged topical or systemic use. The trabecular meshwork (TM) outflow pathway contains several tissues. From upstream to downstream of aqueous humor outflow, the tissues can be divided into proximal outflow tissues including the TM and Schlemm’s canal, as well as distal outflow tissues including the collector channel and aqueous vein. The importance of the distal flow tissue is not clear in glucocorticoid-induced OHT/glaucoma. Therefore, we will study our hypothesis that the distal outflow tissue, like the TM, also responds differentially to glucocorticoids and this response contributes to OHT. Our goal is to elucidate the molecular mechanism of glucocorticoid-induced OHT. We propose two specific aims. SA1. Determine if distal outflow tissues differentially regulate outflow resistance in GIG. Using our new perfusion culture model, we will perfuse paired human donor corneal rims in two study groups. Group a: Both rims will have the TM removed using the Kahook dual blade. One rim will be perfused with 0.1% ethanol (EtOH) as a vehicle control and the fellow rim with 100nM dexamethasone (DEX) for 7-10 days. IOP will be recorded and compared between eyes. At least 50 pairs of corneal rims will be studied. This group is used to determine if the distal outflow tissue is able to cause glucocorticoid-induced OHT. Group b: One rim will be perfused directly, while the fellow rim will have the TM tissue removed and then perfused. Both rims will be treated with DEX for 7-10 days and IOP will be recorded. At least 50 pairs of corneal rims will be studied. This group is used to determine if the distal outflow tissue is as important as the proximal tissue in glucocorticoid- induced OHT. SA2. Compare spatial gene expression between proximal and distal outflow tissues in GIG. Paired corneal rims with or without TM will be perfused with EtOH in one rim and DEX in the fellow rim. Both rims will be used for spatial gene expression studies. Tissues will be fixed and stained with hematoxylin and eosin to identify the outflow structure/region. Identified regions will be mounted on a chip on a slide. The chip contains 5000 bar-coded spots containing oligonucleotides for the capture, amplification, and identification of tissue RNA for RNA sequencing. The resolution of the chip is sufficient to study both proximal and distal outflow tissues. Both responder and non-responder tissues will be studied and compared. The results will be validated using in situ hybridization and immunostaining. Our project is novel because we will use the new corneal rim perfusion culture model and spatial gene expression technology. This study is significant because it will provide a novel treatment target, the distal outflow tissue, for treating glucocorticoid induced glaucoma.