Electron probe X-ray microanalysis of intact pathway for human aqueous humor outflow

Abstract

Intraocular pressure (IOP) is regulated by the resistance to outflow of the eye's aqueous humor. Elevated resistance raises IOP and can cause glaucoma. Despite the importance of outflow resistance, its site and regulation are unclear. The small size, complex geometry, and relative inaccessibility of the outflow pathway have limited study to whole animal, whole eye, or anterior-segment preparations, or isolated cells. We now report measuring elemental contents of the heterogeneous cell types within the intact human trabecular outflow pathway using electron-probe X-ray microanalysis. Baseline contents of Na(+), K(+), Cl(-), and P and volume (monitored as Na+K contents) were comparable to those of epithelial cells previously studied. Elemental contents and volume were altered by ouabain to block Na(+)-K(+)-activated ATPase and by hypotonicity to trigger a regulatory volume decrease (RVD). Previous results with isolated trabecular meshwork (TM) cells had disagreed whether TM cells express an RVD. In the intact tissue, we found that all cells, including TM cells, displayed a regulatory solute release consistent with an RVD. Selective agonists of A(1) and A(2) adenosine receptors (ARs), which exert opposite effects on IOP, produced similar effects on juxtacanalicular (JCT) cells, previously inaccessible to functional study, but not on Schlemm's canal cells that adjoin the JCT. The results obtained with hypotonicity and AR agonists indicate the potential of this approach to dissect physiological mechanisms in an area that is extremely difficult to study functionally and demonstrate the utility of electron microprobe analysis in studying the cellular physiology of the human trabecular outflow pathway in situ.

Fig. 1.

Schematic representation of trabecular outflow pathway for human aqueous humor at lower (A) and higher (B) resolution. The ciliary epithelium covering the surface of the ciliary body (CB) secretes the aqueous humor into the posterior chamber (PC) and thereafter crosses the pupil to enter the anterior chamber. Fluid exits through the uveal trabecular meshwork (uTM) in series with the corneoscleral trabecular meshwork (TM), juxtacanalicular tissue (JCT), Schlemm's canal inner wall (SCI), Schlemm's canal outer wall (SCO), collector channels (CC), and episcleral veins (not shown). EW identifies the external wall contacting the outer wall of Schlemm's canal. Ciliary muscle (CM) fibers attach to the scleral spur (SS) and to the uTM.

Fig. 2.

Unstained freeze-dried tissue section of human trabecular outflow pathway, illustrating the microscopic anatomy.

Fig. 3.

Representative freeze-dried tissue section illustrating identification of cells included in analyses presented in tables. Arrows identify cells SCI and SCO, the JCT, corneoscleral TM, and uTM.

Fig. 4.

Representative electron probe X-ray microanalytic spectra under control (A) and ouabain-exposed (B) conditions.

Fig. 5.

Relationship between cell volume, monitored as (Na+K)/P, and normalized chloride content (Cl/P) for the different cell types of the outflow pathway. Data points are taken from Table 2. The slope of the linear least-squares fit is 1.1 ± 0.2 (P = 0.003, R = 0.925).

Fig. 6.

Bar graphs of normalized Cl⁻ content (A) and normalized cell volume (B), monitored as (Na+K)/P, as functions of cell type and experimental condition. The interrupted horizontal lines present the Cl⁻ contents (A) and volume (B) of SCI cells under control conditions. Cells subjected to hypotonic challenge (Hypo) were incubated in solutions with osmolalities of either 200 or 152 mosmol/kgH₂O. The osmolality of the other incubation solutions was 290 mosmol/kgH₂O.