Toward in vivo two-photon analysis of mouse aqueous outflow structure and function

Abstract

The promise of revolutionary insights into intraocular pressure (IOP) and aqueous humor outflow homeostasis, IOP pathogenesis, and novel therapy offered by engineered mouse models has been hindered by a lack of appropriate tools for studying the aqueous drainage tissues in their original 3-dimensional (3D) environment. Advances in 2-photon excitation fluorescence imaging (TPEF) combined with availability of modalities such as transgenic reporter mice and intravital dyes have placed us on the cusp of unlocking the potential of the mouse model for unearthing insights into aqueous drainage structure and function. Multimodality 2-photon imaging permits high-resolution visualization not only of tissue structural organization but also cells and cellular function. It is possible to dig deeper into understanding the cellular basis of aqueous outflow regulation as the technique integrates analysis of tissue structure, cell biology and physiology in a way that could also lead to fresh insights into human glaucoma. We outline recent novel applications of two-photon imaging to analyze the mouse conventional drainage system in vivo or in whole tissues: (1) collagen second harmonic generation (SHG) identifies the locations of episcleral vessels, intrascleral plexuses, collector channels, and Schlemm's canal in the distal aqueous drainage tract; (2) the prospero homeobox protein 1-green fluorescent protein (GFP) reporter helps locate the inner wall of Schlemm's canal; (3) Calcein AM, siGLO™, the fluorescent reporters m-Tomato and GFP, and coherent anti-Stokes scattering (CARS), are adjuncts to TPEF to identify live cells by their membrane or cytosolic locations; (4) autofluorescence and sulforhodamine-B to identify elastic fibers in the living eye. These tools greatly expand our options for analyzing physiological and pathological processes in the aqueous drainage tissues of live mice as a model of the analogous human system.

Keywords: Aqueous humor outflow; Collector channel; Conventional outflow; Glaucoma; Multiphoton microscopy; Schlemm’s canal; Second harmonic generation.

Figure 1

This figure orientates the reader to the imaging perspective created by deep tissue 2-photon microscopy of the mouse aqueous drainage tissues. A: Light micrograph of a hematoxylin and eosin-stained cryosection from a Balb/c mouse reveals its anatomy. Lens is at the top (internal); cornea to the right (anterior; stroma); choroid to the left (posterior); and sclera and conjunctiva at the bottom (external). Schlemm’s canal (SC) is a space between the trabecular meshwork (TM) and sclera. Intrascleral plexus (ISP) channels are openings in the mid to external sclera. In this instance, the postmortem iris is flush with the corneal endothelium (right). Dashed cyan lines indicate inner and outer limits of the sclera (and corneal stroma to the right) featuring dense collagen. The box indicates the 2P imaging volume at 630X power as represented in panels B–D. B: Mouse TPEF of labeled F-actin (Alexa Fluor 568-conjugated phalloidin; red) and SHG of collagen (cyan) mirrors the organization shown in panel A. The dense collagen is collagen SHG-positive and intertwined collagen fiber bundles are seen in the rotated en face aspect of panel D (see later). F-actin lines ISPs. C: Isosurface volume reconstruction integrating F-actin TPEF (red) and SHG (cyan, flat) signals, and signal voids of channel lumen (darker cyan, contoured). The SHG signal is rendered transparent, revealing the 3D-mapped signal voids representing collector channels (CC) connecting to ISP within the sclera. D: The image volume is rotated obliquely and surface structures cut away in the software to reveal ISP just deep to the ocular surface.

Figure 2

3-dimensional (3D) reconstructions and isosurface maps derived from serial optical sections through the limbal scleral region of live transgenic m-Tomato (membrane-directed td-Tomato) mice. Episcleral veins (ESV), collector channels (CC) and collector channel ostia were easily identified in live mice in which m-Tomato fluorescent reporter expression and second harmonic generation (SHG) imaging were combined. A–D: Transgenic m-Tomato expression (red) was seen in cell membranes revealing the location of an ESV in a scleral groove (A) near the conjunctival epithelium surface (B; red cellular sheet), and intrasceral plexus (ISP), CC (C), and Schlemm’s canal (SC) deeper in the sclera. D: 3D isosurface map of m-Tomato with respect to scleral collagen SHG closely resembles direct 3D reconstruction from optical sections (C). E, F: Isosurface map of SHG (E) and reconstruction of m-Tomato and SHG (F) cropped near the external ocular surface reveals ESVs as channel-like SHG voids (arrows) flanked by m-Tomato-expressing endothelial cells. G, H: 2X magnified views of reconstructions (G) and isosurface maps (H) of m-Tomato and SHG reveal submicron-level detail. Bar=25 µm.

Figure 3

2-photon (2P) navigation of the mouse conventional outflow pathway guided by autofluorescence, collagen second harmonic generation (SHG) and filamentous actin (F-actin) signals. Eyes were enucleated and not fixed (A, D, G, J) or fixed in paraformaldehyde and labeled with Alexa-568-conjugated phalloidin (B, C, E, F, H, I, K, L) and imaged with TPEF for autofluroescence (green; A, D, G, J), SHG (cyan; B, E, H, K), or SHG and F-actin (red; C, F, I, L show merged SHG and F-actin signals). A, B, C: Conjunctival epithelium on the corneoscleral limbal surfce is at the bottom of each panel. The 2P laser passes ‘outside-in’ through the ocular surface structures, then sclera, intrascleral plexuses (ISP), collector channels (CC), Schlemm's canal (SC), and the trabecular meshwork (TM) in succession. ISP, CC, SC, and TM are identifiable by the location and pattern of signal voids in autofluorescence and SHG images at the indicated depths. The signal voids of ISP, CC and SC were continuous through different scleral depths. At the level of the TM shown, a network of linear and branching elastin fibrils surrounding signal voids representing pores was seen by autofluorescence (D) but not SHG (E). The location of cells was indicated by F-actin. Cells associated with the branching fibrils surrounding pores with a diameter > 25um (F). In SC, a pattern of walls and septae as seen by autofluorescence (G) and SHG (H) produced an appearance of loculation. CC were seen in cross-section as oval or circular signal voids with diameters of 15–20 µm and associated with F-actin (L) in autofluorescence (J) and SHG (K, L) images. Bar=25 µm.

Figure 4

Prox1-GFP reporter identifies the inner wall of Schlemm's canal (IWSC) in the mouse. A–F: 3D reconstructions and isosurface mapping (E, F) of Prox1-GFP (green) reporter mouse limbus after TPEF imaging. Prox1-GFP signal manifests as interconnected cells in a monolayer (A) aligned in a circumferential ring (B) just anterior to a scleral spur (SS; C), seen as a condensed ridge of structural collagen by second harmonic generation (SHG; D) and 3D reconstruction. D: Anterior-posterior distance of SC from SS to the anterior limit of Schlemm's canal (SC) was approximately 140 µm. E: Isosurface mapping of SHG (cyan) reveals the SS and a slight trench to the anterior (to the right) indicating the outer wall of SC. F: Isosurface map of Prox1-GFP is merged with SHG (from E), showing Prox1-GFP localization to the region of the IWSC. G–L: Whole mount staining of Prox1-GFP anterior segment with antibodies against lymphatic vessel endothelial hyaluronan receptor (LYVE-1; red) reveals location of the Prox1-GFP positive, LYVE-1 negative IWSC (asterisks) and the Prox1-GFP positive, LYVE-1 positive limbal lymphatic vessels (arrows). G, J: Merge of Prox1-GFP and LYVE-1. H, K: Prox1-GFP only. I, L: LYVE-1 only. J–L: 5X magnifications of regions indicated by opposing arrowheads in panels G–I.

Figure 5

Visualization of live cells and elastin in mouse eyes using siGLO™ and sulforhodamine-B, exogenous fluorescence labels. A–B: projection images from serial optical sections; C: orthogonal reconstruction; D–H: isosurface mapping of the corneoscleral limbus region of C57BL/6 mouse eye after exposure to siGLO™ (red). A: Projection image of sclera combining optical sections located between the vertical dashed lines in panel C showing cell-associated siGLO™ signal. B: SHG signal voids at mid-depth of sclera reveal the location of an intrascleral plexus (ISP). siGLO™ associates with ISP vessels (more clearly seen in C) but also extra-ISP regions of the sclera. C: Orthogonal reconstruction reveals strong siGLO™ in Schlemm's canal (SC) and distal intrascleral channels. D–H: Isosurface maps of siGLO™ (D) and ISP (E) within the sclera, shown as collagen SHG rendered transparent from an outside-in perspective. In the orthogonal perspective, collector channels (CC) are easily appreciated, extending from the ISP (yellow) to the outer wall of SC (blue craggy undulating structure on left; F). The siGLO™ signal is much brighter in cells of the TM and ciliary body (G, H), indicating preferentially labeling of these more proximal drainage structures. In G and H, a slit-like space between the TM (red) and outer wall of SC (red) indicates SC. I–K: Sulforhodamine-B rapidly labels elastin in live mouse eyes, seen here as distinct elastic fibrils with associated stromal and cellular components as the dye also labels cell membranes (I). Near the ocular surface of the corneoscleral limbus (depth of 4 µm) path of an episcleral vessel (ESV) is revealed by lack of sulforhodamine fluorescence surrounded by elastic fibrils and more amorphous labeling. J: Deeper (depth of 44 µm; deep to SC), sulforhodamine-B labels elastic fibers and cells of the TM. Asterisks show fluorescence obstructions caused by pigment (melanin) in the TM. K: Collagen SHG orthogonal reconstruction showing locations (dashed lines) of suflorhodamine-B-labeled optical sections of I and J with respect to SC. Bar=25 µm.