Abstract
Purpose: :
The lipid mediator sphingosine 1-phosphate (S1P) decreases outflow facility in perfused human and porcine eyes. S1P effects have been ascribed to trabecular meshwork (TM) cells, however our recent finding that S1P receptors are expressed by Schlemm's canal (SC) inner wall cells raises the possibility that S1P may be acting directly on SC inner wall (Sumida et al., ARVO 2007). In porcine eyes, S1P dramatically increases giant vacuole density with no obvious changes in the juxtacanalicular (JCT) region (Mettu et al., IOVS, 2004). These observations, along with known effects of S1P on junctional assembly between vascular endothelial cells, motivated us to examine the impact of S1P on SC, TM and JCT morphology in perfused human eyes.
Methods: :
Paired ostensibly normal post mortem human eyes were perfused for 60-90 min at 8 mmHg constant pressure (equivalent to 15 mmHg in vivo) using standard techniques to measure a baseline facility. Anterior chamber contents were exchanged and eyes were further perfused for approximately three hours with 5.0 µM S1P (experimental) or vehicle (control), then perfusion-fixed under pressure. Anterior chamber tissue wedges were cut and either dissected to expose the inner wall of the SC (for immunohistology and scanning electron microscopy (SEM)) or the outflow region was isolated but left intact (for semi-thin sectioning). The tissue was then processed and examined using conventional methods for immunohistology (to label Cadherin-5 and F-actin), light microscopy and SEM.
Results: :
In perfusion studies, baseline outflow facilities were in the normal range (mean = 0.24 µl/min/mmHg; n = 8 pairs), decreasing gradually for 60-90 minutes after S1P administration. The net facility decrease due to S1P was 37 ± 22% (mean ± std. dev; p = 0.003). Semi-thin sections through the outflow region revealed no obvious effects of S1P perfusion on the gross morphology of the SC, TM and JCT, although more subtle structural changes may have been present. In particular, we observed no increase in giant vacuole density. Examination of the inner wall of the SC also showed no obvious S1P effects on Cadherin-5 distribution, however peripheral F-actin appeared to be more highly organized in S1P perfused tissue.
Conclusions: :
Although S1P decreases outflow facility in both human and porcine eyes, morphological observations differ between these species: no gross morphological changes were readily apparent in S1P perfused human tissues, although we did observe some reorganization of peripheral F-actin in SC inner wall cells.
Keywords: cell adhesions/cell junctions • outflow: trabecular meshwork • microscopy: light/fluorescence/immunohistochemistry