Ultrastructural and biochemical evaluation of the porcine anterior chamber perfusion model.

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)-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.

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. 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.

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-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.