Ultrastructural and biochemical evaluation of the porcine anterior chamber perfusion model
Ultrastructural and biochemical evaluation of the porcine anterior chamber perfusion model
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DOI：
10.1167/iovs.05-1393
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发表时间：
2006-05-01
影响因子：
4.4
通讯作者：
Luetjen-Drecoll, Elke
中科院分区：
文献类型：
--
作者：
Bachmann, Bjoern;Birke, Marco;Luetjen-Drecoll, Elke
目的。目的：评价猪前房血流灌注模型，并测试该模型所获得的数据在人体系统中的可转移性。方法：从当地屠宰场获取猪的眼睛，并在死后2小时内进行处理。手术切除晶状体、玻璃体、虹膜和睫状突，切开前房，分别灌流72(40对)或140(2对)小时，或加入10 ng/mL转化生长因子-β(2)。对设备进行连续测量。然后，从所有象限制备小梁网络(TM)标本，并对切片进行形态和免疫组织化学分析。以10对非灌注眼的TM切片作为对照。从TM标本中提取RNA和蛋白质。用Northern和Western印迹法检测αB晶状体蛋白、纤维连接蛋白(FN)、纤溶酶原激活物抑制物(PAI)-1、凝血酶反应蛋白(TSP)-1和结缔组织生长因子(CTGF)的表达。时间上，睫状肌(CM)明显，巩膜沟长而平，巩膜骨刺向虹膜根部延伸。鼻腔CM较薄，沟深，骨刺致密。外TM在巩膜骨刺和角膜之间扩张，覆盖整个圆周。在超微结构水平上，弹力网与筛状TM细胞和房水丛内皮细胞相连。灌流本身对外膜的形态影响很小。房水丛环仍然开放，TM细胞没有坏死或固缩的迹象。在灌注眼中，αB-晶状体蛋白的表达显著增加。用转化生长因子-β(2)灌流72小时后，流出功能减少到中等灌流对照组的60%左右。与推测的引流通路相邻的TM细胞可见粗面内质网(RER)池增大，这是蛋白质合成活跃的标志。αB-晶状体蛋白和FN基因的表达分别增加5倍和3倍。这些蛋白质上调了2.5倍。此外，在人类TM细胞培养实验中，转化生长因子-β(2)上调PAI-1(1.7倍)和TSP-1(1.6倍)蛋白，这两个因素被证明是转化生长因子-β(2)反应的。结论：这些新的超微结构研究表明，猪TM的筛状区和内皮下区域具有与灵长类TM相似的结构。对转化生长因子-β(2)的生化和生理反应与人TM细胞培养和前房灌流中所描述的一致。猪前房灌流模型适用于人体系统。
PURPOSE. To evaluate a porcine anterior chamber perfusion model and to test the transferability of data obtained with this model to the human system.METHODS. 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)-beta(2). 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 alpha B-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-beta 2-perfused anterior segments was examined by Northern and Western blot analyses.RESULTS. 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. alpha B-crystallin expression was significantly increased in perfused eyes. Perfusion with TGF-beta(2) 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 alpha B-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-beta(2) upregulated PAI-1 (1.7-fold) and TSP-1 (1.6-fold) proteins, two factors shown to be TGF-beta(2) responsive in human TM cell culture experiments. CTGF expression was not altered.CONCLUSIONS. 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-beta(2) 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.