A New View of the Human Trabecular Meshwork Using Quick-freeze, Deep-etch Electron Microscopy

Abstract

Conventional transmission electron microscopic (TEM) images of the juxtacanalicular tissue (JCT) region of the trabecular meshwork (TM) show flow passages that are much too large to generate significant outflow resistance. The goal of the current study was to use quick-freeze/deep-etch (QF/DE), a technique that better preserves extracellular matrices, to determine if extracellular matrix material fills all the apparently ‘open-spaces’ that were observed using conventional TEM. Normal adult human eyes were fixed by immersion or under flow at pressures of 15 or 45 mmHg. The TM and the inner wall of Schlemm's canal (SC) were examined using QF/DE and compared to the observations using conventional TEM. The structure of the TM, as seen using QF/DE, showed much greater three dimensional ultrastructural detail than was seen with conventional TEM. Open space was confirmed to be present between the trabecular beams. Although significantly more extracellular matrix was observed in the JCT region using QF/DE than by conventional TEM, some micron-sized open-spaces were still present immediately beneath the inner wall of SC. Consistent with prior reports, the basement membrane of the cells lining the inner wall of SC exhibited discontinuities but the basement membrane as seen by QF/DE was much more elaborate and complex than was evident in conventional TEM of immersion-fixed eyes. This basal lamina became less continuous with increasing perfusion pressure. QF/DE, although difficult and very labor-intensive when used to examine the TM, offers several clear advantages over conventional methods of tissue preparation for ultrastructural study. Although a more complex and less open extracellular matrix structure was seen in the JCT using QF/DE compared with conventional TEM, some open-spaces, similar in size to those seen by TEM, were still observed in this region. The continued presence of such open-spaces in QF/DE images suggests that either the JCT may not generate a significant fraction of outflow resistance in normal eyes or even QF/DE is not sensitive enough to preserve and identify all the extracellular matrix in the JCT region of the TM. SC appears to exhibit a discontinuous basal lamina like lymphatic channels but, like venules, exhibits a wide lumen and continuous endothelium. Because of these features, and the presence of continuous tight junctions that typify continuous capillaries, but neither lymphatic channels nor venules, SC has traditionally been described as a vessel sui generis. Our observations of perfusion-induced changes in the basal lamina of the inner wall of SC suggest that the discontinuous basal lamina underlying the inner wall of SC may not represent the normal expression of the vessel but may simply be a consequence of the way in which giant vacuoles and their pores give rise to outflow.