Bachmann Bjoern, Birke Marco, Kook Daniel, Eichhorn Michael, Lütjen-Drecoll Elke
Department of Anatomy II, Friedrich-Alexander-University, Erlangen, Germany.
Invest Ophthalmol Vis Sci. 2006 May;47(5):2011-20. doi: 10.1167/iovs.05-1393.
To evaluate a porcine anterior chamber perfusion model and to test the transferability of data obtained with this model to the human system.
Porcine eyes were obtained from a local abattoir and processed within 2 hours after death. Anterior chambers of 42 pairs of eyes were dissected with removal of lens, vitreous, iris, and ciliary processes and perfused for 72 (40 pairs) or 140 (2 pairs) hours with medium or medium supplemented with 10 ng/mL transforming growth factor (TGF)-beta2. Facility was continuously measured. Afterward, trabecular meshwork (TM) specimens from all quadrants were prepared, and sections were analyzed morphologically and with immunohistochemical methods. TM sections of 10 nonperfused pairs of eyes were used as the control. RNA and protein was extracted from the TM specimens. Expression of alphaB-crystallin, fibronectin (FN), plasminogen activator inhibitor (PAI)-1, thrombospondin (TSP)-1, and connective tissue growth factor (CTGF) mRNA and protein in medium-perfused and TGF-beta2-perfused anterior segments was examined by Northern and Western blot analyses.
The nonperfused TM showed prominent differences between the temporal and nasal quadrants. Temporally, the ciliary muscle (CM) was pronounced, the scleral sulcus was long and flat, and the scleral spur extended toward the iris root. Nasally, the CM was thin, the sulcus deep, and the spur compact. The outer TM was expanded between the scleral spur and cornea throughout the entire circumference. On the ultrastructural level, the elastic network was connected to the cribriform TM cells and the aqueous plexus endothelium. Perfusion itself had only small effects on the morphology of the outer TM. Aqueous plexus loops remained open, and TM cells showed no signs of necrosis or pyknosis. alphaB-crystallin expression was significantly increased in perfused eyes. Perfusion with TGF-beta2 for 72 hours reduced outflow facility to approximately 60% of that of the medium-perfused control. TM cells adjacent to putative drainage pathways showed enlarged cisterns of rough endoplasmic reticulum (rER), a sign of active protein synthesis. Expression of alphaB-crystallin and FN mRNA were elevated by factors of 5 and 3, respectively. The proteins were upregulated by a factor of 2.5. In addition, TGF-beta2 upregulated PAI-1 (1.7-fold) and TSP-1 (1.6-fold) proteins, two factors shown to be TGF-beta2 responsive in human TM cell culture experiments. CTGF expression was not altered.
These new ultrastructural investigations indicate that the cribriform and subendothelial regions of the porcine TM have an architecture similar to that of the primate TM. The biochemical and physiological response to TGF-beta2 was identical with that described in human TM cell culture and anterior chamber perfusion. The porcine anterior chamber perfusion model is valid for the human system.
评估猪眼前房灌注模型，并测试该模型所获数据向人体系统的可转移性。
从当地屠宰场获取猪眼，并在死后2小时内进行处理。解剖42对猪眼的前房，去除晶状体、玻璃体、虹膜和睫状体，并使用培养基或添加10 ng/mL转化生长因子（TGF）-β2的培养基灌注72小时（40对）或140小时（2对）。持续测量房水流畅度。之后，制备所有象限的小梁网（TM）标本，并通过形态学和免疫组织化学方法对切片进行分析。10对未灌注眼的TM切片用作对照。从TM标本中提取RNA和蛋白质。通过Northern和Western印迹分析检测αB-晶状体蛋白、纤连蛋白（FN）、纤溶酶原激活物抑制剂（PAI）-1、血小板反应蛋白（TSP）-1和结缔组织生长因子（CTGF）mRNA及蛋白质在培养基灌注和TGF-β2灌注的眼前节中的表达。
未灌注的TM在颞侧和鼻侧象限之间存在显著差异。在颞侧，睫状肌（CM）明显，巩膜沟长且平坦，巩膜突向虹膜根部延伸。在鼻侧，CM薄，沟深，突紧密。整个圆周上，外侧TM在巩膜突和角膜之间扩展。在超微结构水平上，弹性网络与筛状TM细胞和房水丛内皮相连。灌注本身对外侧TM的形态影响较小。房水丛环保持开放，TM细胞未显示坏死或固缩迹象。灌注眼的αB-晶状体蛋白表达显著增加。用TGF-β2灌注72小时使房水流畅度降低至培养基灌注对照组的约60%。与假定引流途径相邻的TM细胞显示粗面内质网（rER）池扩大，这是活跃蛋白质合成的迹象。αB-晶状体蛋白和FN mRNA的表达分别升高了5倍和3倍。蛋白质上调了2.5倍。此外，TGF-β2上调了PAI-1（1.7倍）和TSP-1（1.6倍）蛋白，这两种因子在人TM细胞培养实验中显示对TGF-β2有反应。CTGF表达未改变。
这些新的超微结构研究表明，猪TM的筛状和内皮下区域具有与灵长类TM相似的结构。对TGF-β2的生化和生理反应与在人TM细胞培养和眼前房灌注中描述的相同。猪眼前房灌注模型对人体系统有效。
