A Porcine Anterior Segment Perfusion and Transduction Model With Direct Visualization of the Trabecular Meshwork.
A Porcine Anterior Segment Perfusion and Transduction Model With Direct Visualization of the Trabecular Meshwork.
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DOI：
10.1167/iovs.15-18125
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发表时间：
2016-03
影响因子：
4.4
通讯作者：
Loewen NA
中科院分区：
文献类型：
--
作者：
Loewen RT;Roy P;Park DB;Jensen A;Scott G;Cohen-Karni D;Fautsch MP;Schuman JS;Loewen NA
建立一个一致的和负担得起的，高质量的猪眼前节灌注和转导模型，允许直接可视化的小梁网。在死亡后2小时内，通过移除透镜和葡萄膜并固定在专门设计的薄底培养皿中培养猪眼前节，以允许直接观察小梁网，且变形最小。22个控制眼睛（CO）与一个恒定的流量进行了比较，8重力灌注的眼睛（COgr，15毫米汞柱）。我们使用表达eGFP的猫免疫缺陷病毒（FIV）载体GINSIN以每只眼睛108个转导单位（TU）（GINSIN_8，n = 8）和107个TU（GINSIN_7，n = 8）建立了向TM的基因递送。在获得组织学之前评估表达14天。猪眼是一致和高质量眼前节培养的可靠来源，失败率低（12%）。与重力灌注COgr（插补3.7 ± 1.6 μL/min）相比，对照眼在3 μL/min的固定泵灌注下的眼内压（IOP）为15.8 ± 1.9 mm Hg。载体GINSIN_8眼在转导后IOP一过性升高44%，48小时后消退;在GINSIN_7眼中未观察到这一情况。GINSIN_8眼的表达高于GINSIN_7眼。小梁网结构保存完好。与以前使用的人供体眼相比，这种廉价的猪眼前节灌注模型具有足够的，可重复的高质量，以开发TM生物工程的策略。可直接观察到小梁网。尽管存在显著的解剖学差异，但转导的作用复制了先前探索的人、猫和啮齿动物模型的主要方面。
To establish a consistent and affordable, high quality porcine anterior segment perfusion and transduction model that allows direct visualization of the trabecular meshwork. Porcine anterior segments were cultured within 2 hours of death by removing lens and uvea and securing in a specially designed petri dish with a thin bottom to allow direct visualization of the trabecular meshwork with minimal distortion. Twenty-two control eyes (CO) with a constant flow rate were compared to eight gravity perfused eyes (COgr, 15 mm Hg). We established gene delivery to the TM using eGFP expressing feline immunodeficiency virus (FIV) vector GINSIN at 108 transducing units (TU) per eye (GINSIN_8, n = 8) and 107 TU (GINSIN_7, n = 8). Expression was assessed for 14 days before histology was obtained. Pig eyes were a reliable source for consistent and high quality anterior segment cultures with a low failure rate of 12%. Control eyes had an intraocular pressure (IOP) of 15.8 ± 1.9 mm Hg at fixed pump perfusion with 3 μL/min compared to gravity perfused COgr with imputed 3.7 ± 1.6 μL/min. Vector GINSIN_8 eyes experienced a transient posttransduction IOP increase of 44% that resolved at 48 hours; this was not observed in GINSIN_7 eyes. Expression was higher in GINSIN_8 than in GINSIN_7 eyes. Trabecular meshwork architecture was well preserved. Compared with previously used human donor eyes, this inexpensive porcine anterior segment perfusion model is of sufficient, repeatable high quality to develop strategies of TM bioengineering. Trabecular meshwork could be observed directly. Despite significant anatomic differences, effects of transduction replicate the main aspects of previously explored human, feline and rodent models.