Department of Pharmacology and Therapeutics, Faculty of Medicine of the University of Porto, Portugal; Department of Ophthalmology, Hospital S. João, Porto, Portugal; Center for Drug Discovery and Innovative Medicines, University of Porto, Portugal.
Department of Ophthalmology, Hospital S. João, Porto, Portugal.
Exp Eye Res. 2018 Mar;168:107-114. doi: 10.1016/j.exer.2018.01.008. Epub 2018 Jan 13.
We set out to demonstrate that the major source of corneal catecholamines is its neuronal release from intrinsic sympathetic nerves rather than circulating or non-neuronal local production. Three concentric segments (central, intermediate, peripheral) were obtained by double trephination (9.5-7.25 mm) performed on corneas harvested from 3 to 4 month old rabbits and human corneas rejected for transplantation, along with aqueous humour, full iris tissue and blood samples. Endogenous catecholamines were quantified by high pressure liquid chromatography with electrochemical detection (HPLC-ED), and comparison with the uptake of radio-labelled noradrenaline (3H-NA) before and after incubation with cocaine was performed. Results are means ± SEM. Ratios between enzymatic end products and their substrates were calculated. ANOVA was used for statistical analysis. Catecholamine levels were found to be about one log unit lower in the human cornea than in the rabbit cornea. In the rabbit, dopamine (DA), noradrenaline (NA) and adrenaline (AD) were identified by HPLC-ED in all corneal segments, whilst in the human cornea NA was identified only in the intermediate and peripheral corneal segments, and no AD was found. In the iris and aqueous humour only DA and NA were present. A concentration gradient for NA decreasing from the periphery to the centre of the cornea was identified in both species (NA/DA ratio higher than 1 in the periphery; low AD/NA ratio in all corneal segments), but not for DA or AD. After incubation with 3H-NA all corneal segments and iris tissue showed loading with the aforementioned gradient being reproduced, and a decrease in 3H-NA loading after cocaine was significant only in the peripheral corneal segment and in the iris of both species. Reduction in 3H-NA loading after incubation with cocaine shows that NA in the cornea is mostly of neuronal origin and demonstrates the presence of functional sympathetic nerves (also expectedly found in the iris); the existence of a gradient both for 3H-NA loading and loading reduction after cocaine points to a higher density of fibres in the peripheral cornea.
我们旨在证明角膜儿茶酚胺的主要来源是其内在交感神经的神经元释放，而不是循环或非神经元局部产生。通过在取自 3 至 4 个月大的兔子和因移植而被拒绝的人角膜上进行双环钻术（9.5-7.25mm），获得三个同心段（中央、中间、周边），以及房水、全虹膜组织和血液样本。通过高效液相色谱电化学检测（HPLC-ED）定量内源性儿茶酚胺，并在孵育前后与可卡因一起比较放射性标记的去甲肾上腺素（3H-NA）的摄取。结果表示为平均值±SEM。计算酶终产物与其底物之间的比值。采用方差分析进行统计分析。发现人角膜中的儿茶酚胺水平比兔角膜中的儿茶酚胺水平低一个对数单位。在兔子中，通过 HPLC-ED 在所有角膜段中鉴定出多巴胺（DA）、去甲肾上腺素（NA）和肾上腺素（AD），而在人角膜中仅在中间和周边角膜段中鉴定出 NA，并且未发现 AD。在虹膜和房水中仅存在 DA 和 NA。在两种物种中都确定了从周边到角膜中心的 NA 浓度梯度降低（周边的 NA/DA 比值高于 1；所有角膜段的低 AD/NA 比值），但 DA 或 AD 则不然。用 3H-NA 孵育后，所有角膜段和虹膜组织均显示出上述梯度的负载，并且只有在外周角膜段和两种物种的虹膜中，在用可卡因孵育后 3H-NA 负载的减少才具有统计学意义。在用可卡因孵育后 3H-NA 负载的减少表明角膜中的 NA 主要来自神经元，并证明存在功能性交感神经（也预期存在于虹膜中）；3H-NA 负载和可卡因孵育后负载减少的梯度都表明周边角膜的纤维密度更高。
