The structure of the trabecular meshwork, its connections to the ciliary muscle, and the effect of pilocarpine on outflow facility in mice

Abstract

Purpose: To determine the connections between the ciliary muscle (CM), trabecular meshwork (TM), and Schlemm's canal (SC) and their innervations that allows CM contraction (by pilocarpine) to influence conventional outflow in mice.

Methods: Sequential sections and whole mounts of murine corneoscleral angles were stained for elastin, α-smooth muscle actin (αSMA), vesicular acetylcholine transporter (VAChT), neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH). Elastic (EL) fibers between the CM, TM, and SC were examined in ultrathin, sequential sections from different planes. The effect of pilocarpine (100 μM) on conventional outflow facility was measured by perfusion of enucleated mouse eyes.

Results: The mouse TM contains a three-dimensional (3D) net of EL fibers connecting the inner wall of SC to the cornea anteriorly, the ciliary body (CB) internally and the choroid and CM posteriorly. The CM bifurcates near the posterior TM, extending outer tendons to the juxtacanalicular tissue and inner wall of SC and internal connections to the lamellated TM and CB. Ciliary muscle and lamellated TM cells stain with αSMA and are innervated by VAChT-containing nerve fibers, without TH, VIP, or nNOS. Pilocarpine doubled outflow facility.

Conclusions: Mouse eyes resemble primate eyes not only by their well developed SC and TM, but also by their 3D EL net tethering together the TM and SC inner wall and by the tendinous insertion of the CM into this net. The increase in outflow facility following cholinergic stimulation in mice, as in primates, supports using mice for studies of aqueous humor dynamics and glaucoma.

Keywords: aqueous humor; ciliary muscle; glaucoma; pilocarpine; trabecular meshwork.

Figure 1

Conventional outflow pathway of the mouse eye, showing the TM, SC, and connections with the CM. (A) Sagittal semithin section stained with toluidine blue (C57BL/6 mouse). The CB apposes nearly two-thirds of the internal TM surface. Some of the TM lamellae attach to the pectinate ligament (PL) spanning between the peripheral cornea and iris root. (B) Histologic sagittal section showing αSMA labeling in the CM extending into the TM where there is intense labeling throughout the entire juxtacanalicular tissue and lamellated TM (C57BL/6 mouse). The internal and external branches of the CM are indicated. Red blood cells are visible within SC lumen, but are absent from the juxtacanalicular tissue and TM. (C) Whole mount of αSMA labeling showing the anterior-posterior extent of the compact CM from posterior tips (arrows) to the posterior margin of the TM, making occasional connections to the TM (arrowheads). Intense, but incomplete, αSMA staining is also present within the circularly arranged TM, which due to the unlabeled intertrabecular spaces can be distinguished based on its sponge-like appearance from the compact CM (BALB/c mouse). (D) Transmission electron micrograph near the posterior end of SC showing the branching of CM cells (arrow) that are surrounded by a continuous basement membrane. Cell processes connect CM cells to one another. Outer CM cells extend elastic (EL) fiber tendons (arrowhead) that make connections with the inner wall endothelium of SC, and the tendons and CM cells themselves connect to the EL fibers in the juxtacanalicular tissue. The CM is highly innervated by nerve fibers (N; C57BL/6 mouse).

Figure 2

Regional differences in the conventional outflow pathway of the mouse eye. (A) Sagittal semithin section stained for toluidine blue (BALB/c, temporal quadrant). Schlemm's Canal and outer TM are expanded, and the CM is relatively thick compared with Figure 1A. The posterior muscle tips (arrow) extend into the choroid underlying the peripheral retina (R), which is artificially damaged in the section. (B) Histologic sagittal section showing αSMA labeling in the small CM extending along the internal face of the lamellated TM (C57BL/6, nasal quadrant). In contrast to Figure 1B, the αSMA-positive region of the TM does not cover the entire extent of SC, and there is no staining of the outer TM or juxtacanalicular tissue in this section (arrowhead). (C) Whole mount of the CM and TM showing that where the anterior ciliary artery (CA) passes through the sclera into the uvea, there is no αSMA labeling in the CM region (BALB/c, superior quadrant). The TM shows nonuniform αSMA staining. (D) Electron micrograph of a sagittal section through the posterior end of SC and TM in a region near a CA (C57BL/6, superior quadrant). In the area where in other parts of the circumference CM cells are typically present (cf. Fig. 1D), CM cells are absent and there are only connective tissue beams (arrows) continuous with the central core of the TM lamellae.

Figure 3

Elastic fiber net within the conventional outflow pathway of mouse eyes. (A) Histologic sagittal section stained with resorcin-fuchsin for elastin (BALB/c, nasal quadrant). The scleral sulcus is surrounded by stained EL fibers (purple) along the outer wall of SC and in the TM. In this nasal quadrant, there is a well-developed SS containing numerous EL fibers. Purple-stained tendons of the outer CM insert into the SS (arrow). At the anterior surface of the CB stroma, the TM lamellae are cut in a somewhat oblique plane showing EL fibers (arrowheads) extending outwardly from the CB to outer TM. The EL fiber staining is continuous between the anterior TM, pectinate ligament and peripheral cornea (CO). (B) Oblique histologic section through the CB and TM stained with resorcin-fuchsin showing a dense net of EL fibers stretching between the CB and inner wall of SC (C57BL/6 mouse). Arrows indicate nuclei of SC endothelial cells along the inner wall. (C) Whole mount of TM, choroid, and remnants of CM muscle tips (dark areas) stained with resorcin-fuchsin showing longitudinal EL fibers of the choroid and tendons of the CM extending into and merging with the circumferential EL fibers of the TM (BALB/c mouse). Arrows indicate EL fibers and tendons bending into the EL net of the TM. (D) Tangential section through the TM and CM stained with resorcin-fuchsin and picric acid (to stain CM fibers yellow) showing EL tendons (arrows) extending anteriorly from the CM to bend and insert into the EL fiber net of the TM (arrowheads) or into the EL fibers of the CO (C57BL/6 mouse). S, Sclera.

Figure 4

Ultrastructural images of the connections between the EL fiber net and inner wall endothelium of SC and ciliary muscle (CM). (A) Sagittal section through the posterior juxtacanalicular tissue showing that the anterior tips of the CM cells, identified by their complete basement membrane, form fine fibrillar tendons (arrows) that connect to the TM cells in the outer juxtacanalicular tissue, identified by their incomplete basement membrane (C57BL/6 mouse). (B) Sagittal section through the posterior juxtacanalicular tissue and SC showing CM cells, extending toward the inner wall endothelium of the posterior SC (BALB/c mouse). Inset: higher magnification of the CM cell forming a tendon (arrow) that extends toward a juxtacanalicular cell that exhibits significant nuclear deformation, likely resulting from the contractile force of the CM cell. (C) Tangential section through the inner wall endothelium of SC and juxtacanalicular tissue showing numerous EL fibers that make direct connections to SC cells (BALB/c mouse). (D) Tangential section through the inner wall at higher magnification showing direct connections between EL fibers and SC cells (arrowhead) or their basement membrane (arrow; BALB/c mouse).

Figure 5

Whole mounts of the CM and TM of BALB/c mice analyzed with antibodies against αSMA (red) and vesicular acetylcholine transporter (VAChT) or TH (green). (A) There are several VAChT immunoreactive (IR) nerve fibers and terminals along the CM (arrows). (B) Numerous VAChT-IR nerves and terminals (arrows) are also present in the TM. (C) A large TH-IR nerve bundle (arrow) passes through the CM, but there are no reactive nerve varicosities along either CM or TM cells. Single varicosities are observed along small vessels in the CM. (D) The same whole mount as shown in (C), but focused deeper into the episclera, where the episcleral vessels (arrows) are surrounded by TH-IR nerve varicosities.

Figure 6

The flow rate (Q) measured at each perfusion pressure (P) for enucleated mouse eyes with or without 100 μM pilocarpine (Pilo). Pilocarpine significantly increases Q for each P and increases 2-fold the slope of the Q versus P relationship, representing a 2-fold increase in conventional outflow facility (C57BL/6 mice).

Figure 7

A schematic illustration of the architecture of the conventional outflow pathway in mouse eyes, showing the relationship between the CM, TM, and inner wall (IW) of SC. The CM bifurcates into internal and external branches. An EL fiber net is present within the TM and juxtacanalicular tissue, and tendons of the external CM and the EL fibers of the choroid bend to insert into the juxtacanalicular EL net or connect directly to the IW endothelium. The internal CM makes connections to the lamellated TM that is continuous with the CB stroma. Elastic fibers bridge between the lamellated TM and juxtacanalicular tissue, and αSMA-labeled cells (pink) are present within the lamellated TM and occasionally in the juxtacanalicular tissue.