Abstract
Abstract: :
Purpose: In vitro perfusion is widely used to analyze outflow facility. However, limited information is available concerning the viability of the tissue preparations in these in vitro experiments. We evaluated the length of time post mortem that bovine eye preparations can be used reliably to measure outflow facility, in terms of stability of the facility of outflow, smooth muscle constriction capacity, and viability of the corneal epithelial and endothelial cells, trabecular meshwork, and ciliary processes. Methods: Eyes were delivered from the abattoir in cold saline within 3 h of death. The posterior pole (1/4), retina, vitreous, and lens were removed immediately and the segment was fixed on a perfusion chamber. Some eyes (fresh) were perfused immediately with oxygenated DMEM + PSA solution at 34°C at a perfusion pressure of 11 mmHg for 24 h. Fluid flow, temperature, and pressure were monitored electronically. Other eyes were perfused with oxygenated DMEM+PSA at 11 mmHg for 24, 48, 72, or 144 hours at 4°C, after which outflow facility was measured at 34°C for 24 h. After perfusion at 34°C, iris dilator muscle function was tested with 6.0% pilocarpine, then with 2.5 % adrenaline, and the change in pupillary area was analyzed. Finally thin tissue blocks were incubated in H-342 solution to analyze cellular viability under a fluorescence microscope at 350/460 nm. Results: Outflow facility in the fresh eyes was 0.81±0.03 µl/min/mmHg (n=10) at 34°C. After 24 h of perfusion at 4°C, outflow facility was significantly lower (0.67 ±0.03; n=4; P<0.03) vs the fresh eyes. The 48-h value (0.79 ± 0.03; n=6) was not significantly different from the 24-h value, but the 72-h value (0.84 ± 0.03; n=4) was significantly greater than the 24-h value (P<0.02). The 144-h value (1.50 ± 0.05; n=3) was significantly greater than all previous values (P<0.0001). In the fresh eyes, pilocarpine constricted the pupillary area by 34.1%; adrenaline dilated the pupillary area 23.2 % from the constricted state. Corresponding changes after 144 h of perfusion at 4°C were 19.9% constriction and 75.6% dilation. Cellular viability after 144 h of perfusion at 4°C showed only slight degradation vs fresh samples. Conclusions: Facility of outflow changes significantly during perfusion at 4°C for up to 144 h (possibly as a result of protein release leading to changes in the resistance of the outflow pathways), whereas both pupillary function and cellular viability remain nearly normal. Therefore, outflow studies should be performed with eyes that are as fresh as possible (or at least of the same post mortem age) to avoid the confounding effects of long-term perfusion changes in the experimental results.
Keywords: outflow: trabecular meshwork • pupil • immunohistochemistry