The Aqueous Humor Outflow Pathway of Zebrafish

Abstract

purpose. The structures of the ocular anterior segment responsible for aqueous humor secretion and absorption have been well characterized in mammals. However, the underlying molecular and cellular mechanisms that regulate aqueous humor flow have remained elusive. Experimental analysis in Danio rerio, the zebrafish, is providing mechanistic insights into many cellular processes relevant to normal human physiology and disease. To facilitate studies on the molecular and cellular mechanisms of aqueous humor dynamics using this species, the authors have characterized the anatomy of aqueous secretion and outflow in adult zebrafish eyes.

methods. Analyses by light and transmission electron microscopy, coupled with molecular tracers of fluid flow, were used to identify and study the sites of aqueous humor secretion and absorption in adult zebrafish eyes.

results. Zebrafish eyes show aqueous humor secretion primarily from the dorsal ciliary region and outflow through a ventral canalicular network that connects with an aqueous plexus and veins of the choroidal rete.

conclusions. Vectorial flow of zebrafish aqueous humor is in contrast to that in mammals in which secretion and absorption of aqueous humor are circumferential around and through the iridocorneal angle. However, local anatomy and ultrastructure of the tissues and cells specialized for aqueous humor dynamics in zebrafish show conservation with that of mammals. These observations suggest that zebrafish can serve as a useful genetic model to help understand the regulation and cellular basis of normal and abnormal aqueous humor dynamics in humans.

In the ocular anterior segment of vertebrates, the production and outflow of aqueous humor are important determinants of intraocular pressure and are required to maintain the proper shape and optical parameters of the eye. Aqueous humor flow also functions to circulate nutrients and to remove potentially harmful metabolites and cellular debris from the eye. In mammals, aqueous humor is produced circumferentially from the epithelia of ciliary processes. Mechanistically this is achieved by diffusion, ultrafiltration, and active secretion of solutes and water. ^{1 2 3 4} Pigmented and nonpigmented ciliary epithelia function as a syncytium that transports and pumps ions and bioactive proteins into the anterior chamber. ^{5 6} Water from ciliary body vasculature is then moved by osmotic forces across the epithelia and into the posterior chamber. Aquaporins expressed by the ciliary epithelium can also directly transport water to the posterior chamber. ⁷  

Aqueous humor then flows through the pupil into the anterior chamber and drains primarily through specialized structures, which are also organized circumferentially, at the iridocorneal angle. Specifically, the humor filters through the corneoscleral meshwork and trabecular meshwork into Schlemm’s canal. The trabecular meshwork is composed of a uveal layer of endothelial lined, collagen- and elastin-rich trabecular beams covered by endothelial-like trabecular cells and a loose network of extracellular matrix referred to as the juxtacanalicular connective tissue. ^{8 9 10} The juxtacanalicular connective tissue has a complex organization with open areas filled with a glycoproteinaceous extracellular matrix. ^{11 12 13 14 15} Aqueous humor percolates through the trabecular meshwork and ultimately traverses the inner wall of Schlemm’s canal. Aqueous humor then flows from Schlemm’s canal into collector channels of the episcleral venous system, which returns the aqueous to the circulation. The major resistance for fluid drainage is generally thought to lie within the juxtacanalicular connective tissue or the inner wall of Schlemm’s canal, or both. ^{16 17 18 19 20 21} However, unlike the production of aqueous humor, the molecular and cellular mechanisms of aqueous humor outflow are a matter of debate and are not well understood. Understanding the mechanism of aqueous humor outflow has medical significance because defects in outflow are the most common cause of elevated intraocular pressure. ⁴  

Elevated intraocular pressure is a major risk factor for the glaucomas, a group of progressive and blinding diseases of the optic nerve. Therefore, we have characterized the anatomy associated with aqueous humor dynamics in zebrafish. The zebrafish experimental system has emerged as a powerful model for insights into the genetic basis and molecular and cellular mechanisms of normal physiology and disease. However, recent published observations suggest that the anterior segment anatomy of zebrafish varies from that of mammals. ^{22 23 24 25 26} In the present study, we use aqueous humor tracer experiments coupled with localized anatomic characterizations to investigate the aqueous humor outflow pathway of the zebrafish eye. 

Methods

Specimens

Wild-type zebrafish (Danio rerio) of the AB/AB and LF/LF backgrounds were reared under standard conditions with a light cycle of 14 hours light/10 hours dark. No differences in anterior segment anatomy were observed between these two strains. Specimens were collected between 9 to 14 months after fertilization. Before experimental manipulation or tissue fixation, fish were anesthetized in 0.2 mg/mL ethyl 3-aminobenzoate methane sulfonate (tricane). All experiments were performed in compliance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. 

Light Microscopy

Fish tissue was fixed in primary fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 3% sucrose, 0.06% phosphate buffer, pH 7.4) at 4°C for at least 18 hours. Tissue was washed in 0.1 M phosphate-buffered saline (PBS), dehydrated through an ethanol series and propylene oxide, and infiltrated with resin mixture (EMbed-812/Araldyte, Electron Microscopy Sciences, Hatfield, PA). Transverse semithin (1 μm) plastic sections were cut with a glass knife on a microtome (JB4; Thermo Fisher Scientific, Waltham, MA). Serial sections were collected from near the central retina until the ventral canalicular network was first apparent, as indicated by a break in the ventral iris pigment epithelium. After heat fixation to glass slides, sections were stained with 1% toluidine blue in 1% Borax buffer. Images were captured using a digital color camera (Coolpix 995; Nikon, Tokyo, Japan) mounted on a compound microscope (E800; Nikon) with a 60× oil-emersion objective. Serial sectioning through the ventral canalicular network was carried out on 10 eyes. 

Transmission Electron Microscopy

Transmission electron microscopy (TEM) of fish eyes was carried out similarly to that described for the mouse. ¹⁴ Fish were fixed in primary fixative and were washed as for light microscopy. Specimens were then postfixed with 1% osmium tetroxide on ice for 1 hour to preserve the membranes. Tissue was dehydrated through a methanol series and acetonitrile and infiltrated with resin mixture (EMbed-812/Araldyte; Electron Microscopy Sciences). Ultrathin sections (60–70 nm) were collected on coated grids and stained with uranyl acetate and lead citrate for contrast. Images were captured with a transmission electron microscope (H600; Hitachi, Yokohama, Japan). TEM was conducted on six eyes. 

Ophthalmic Artery Injections

Fish were anesthetized as described until they were unconscious and no longer responded to physical stimuli. Once anesthetized, an approximately 2-cm incision was made on the ventral surface of the fish superficial to the heart and gut. The fish were further dissected to expose the ophthalmic artery. The ophthalmic artery enters the dorsoposterior aspect of the eye parallel to the optic nerve. After exposing the ophthalmic artery, approximately 1 μL lysine-fixable, 3-kDa, biotinylated dextran (40 mg/mL) (D7135; Molecular Probes, Invitrogen, Carlsbad, CA) was injected into the artery. The dextran solution was injected with a glass capillary pulled to an approximately 5-μm tip and delivered through a syringe driver. At either 2 or 10 minutes after injection, eyes were enucleated and fixed in 4% paraformaldehyde and subsequently processed for streptavidin-peroxidase binding and reactivity. For each time point, six fish were assessed by ophthalmic artery tracer injections. 

Intraocular Injections

Fish were anesthetized as described until they were unconscious and no longer responded to physical stimuli. Once anesthetized, 500 nL various aqueous humor tracers were injected into the anterior chamber over a period of 5 minutes so as not to damage the anterior segment tissue and to minimize pressure changes. The following molecular tracers were used in these experiments: lysine-fixable, 3-kDa, rhodamine-conjugated dextran (40 mg/mL; D3308; Molecular Probes); purified type VI horseradish peroxidase (HRP; 25 mg/mL; P8375; Sigma, St. Louis, MO); and 10 nm gold-BSA (1:4 diluted stock with PBS; catalog no. 25486; Electron Microscopy Sciences). The solutions were injected with a glass capillary pulled to an approximately 10-μm tip and delivered through a manual syringe driver. Epifluorescence microscopy was used to follow the dynamics of rhodamine-conjugated dextran in eyes from living, anesthetized specimens. For specimens injected with HRP, the heads of the fish were removed at prescribed intervals and fixed overnight in primary fixative. The heads were then bisected and prepared for peroxidase staining by incubation of the tissue in 0.5 mg/mL 3,3′-diaminobenzidine + 0.003% H₂O₂ buffered in PBS. ²⁷ These eyes and those injected with 10 nm gold-BSA were further processed for TEM, as described. For each experiment, at least six eyes were assessed. 

Results

Several features of the zebrafish eye suggest a unique system for aqueous humor circulation (Fig. 1A[and see 2 3 4 Fig. 5 ]). Unlike mammals, the zebrafish eye lacks ciliary processes and also lacks a circumferential organization of trabecular meshwork and ouflow channels. Instead, circumferential at the iridocorneal angle of teleosts is a hypertrophied-appearing structure called the annular ligament. ^{23 25 26 28 29} A recent study has suggested the annular ligament of zebrafish is equivalent to the mammalian trabecular meshwork. ²⁶ Although the morphology of the annular ligament differs significantly from that of mammalian iridocorneal angle specialization, the circumferential nature and location of this structure is consistent with this notion. However, experimental analysis presented here indicates this in not the case. 

Previous studies of the developing anterior segment of zebrafish showed that the dorsal ciliary epithelium, despite lacking processes, appears specialized for aqueous humor secretion. ²³ At the ventral iridocorneal angle, a focal canalicular network suggestive of an aqueous outflow pathway was found. In adult zebrafish, these dorsal and ventral features were maintained. Ultrastructurally, the dorsal ciliary epithelium is composed of apical-apical–facing pigmented and nonpigmented cells coupled by adherens and tight junctions (Fig. 1B) . The nonpigmented epithelium shows numerous vesicles and intracellular membranous infoldings, similar to those of mammalian secretory ciliary epithelium. ^{5 27} By light microscopy the anterior portion of the ventral canalicular network (the iridocorneal canal) appears as an endothelium separating the argentea and stroma of the iris from the annular ligament (Fig. 1C) . The posterior portion of the ventral canalicular network (the ciliary canal) is distinguished by a break between the retinal pigment epithelium and the pigmented ciliary epithelium. The ciliary part of the canalicular network and parts of the angular aqueous plexus fill the space between the breaks in the pigmented epithelia (Figs. 1C 1D) . After fixation, the angular aqueous plexus is often filled with blood cells. In addition, by light microscopy, this region of the ventral angle is noted by the presence of a thumblike structure underlying and associated with the ventral iris (Fig. 1C) . In other species of fish and amphibians, this structure—a muscular tissue that functions to move the lens during accommodation—is called the retractor lentis. ²⁸ However, in zebrafish the retractor lentis may be vestigial because there is no evidence for lens accommodation in this species. ³⁰  

To investigate the finer morphology of the ventral canalicular network, we analyzed this region by TEM and found the canalicular network is lined by an endothelium 2 to 4 cells thick. Endothelial cells lining the canals and openings show numerous clathrin-coated pits and endocytic vesicles (Figs. 1F 1G) . Overall, the endothelium is highly complex in organization such that thin sections give the appearance of a topographical map because of the plasma membranes of the interdigitating cells (Fig. 1H) . Electron-dense junctional complexes couple the interdigitated endothelial cells (Figs. 1H 1I) . At the opening of the canalicular network at the ciliary and iridocorneal regions is loosely organized juxtacanalicular connective tissue (Figs. 1D 1J) . A tortuous canalicular network formed by the spaces between endothelial cells can be traced from the iridocorneal angle to the angular aqueous plexus that resides between the ventral scleral ossicle and the rim of the neural retina (Figs. 1J 1K 1L 1M) . Serial sectioning indicated that the width of the canalicular network typically measured less than 75 μm. Endothelial cells lining the opening of the canalicular network at both the ciliary region and the iridocorneal angle, as well as endothelial cells adjacent to the angular aqueous plexus at the termination of the canalicular network, show parachuting morphologies and giant vacuoles. Their morphologies are similar to those of mammalian endothelial cells lining Schlemm’s canal and arachnoid cells lining the ventricles of the central nervous system (Fig. 1M) . ^{31 32} TEM also revealed the angular aqueous plexus is continuous with vessels of the choroidal rete. Cumulatively, the anatomic structure suggests this region as the outflow pathway for aqueous humor in zebrafish. 

To more directly investigate the routes of aqueous humor movement in zebrafish, we injected 3 kDa biotinylated dextran into the ophthalmic artery of living, anesthetized zebrafish and fixed these specimens 2 or 10 minutes after injection. The ophthalmic artery directly feeds the ciliary vessels that supply aqueous humor in mammals, and this sized dextran is small enough to be secreted with aqueous humor. ^{6 27 33} After injections into the ophthalmic artery, eyes were enucleated and processed as whole tissues for the location of the 3-kDa dextran using streptavidin coupled to horseradish peroxidase (HRP). In the presence of 3,3′-diaminobenzidine, HRP produces a reddish-brown precipitate. Within 2 minutes of injection into the ophthalmic artery, 3 kDa dextran could be detected throughout the anterior chamber, with enrichment in the dorsal and the ventral regions of the eye (Fig. 2A) . By 10 minutes after a single bolus injection, HRP reactivity was found only in the ventral quadrant of the eye, with the most intense staining at the opening of the ventral canalicular network (Fig. 2B) . These data are consistent with morphologic findings suggesting that aqueous humor is produced in the dorsal hemisphere and leaves the anterior segment of the eye through the ventral canalicular network. 

In a related set of experiments, we injected 3 kDa rhodamine-conjugated dextran directly into the ocular anterior segments of anesthetized zebrafish and observed the movement of this tracer by fluorescence microscopy (Figs. 2C 2D) . To minimize changes in IOP induced by fluid injection, 100 nL/min of 3 kDa dextran solution was delivered with a manual syringe driver over a period of 5 minutes. Anesthetized zebrafish rested horizontally throughout the experiment so that gravitational forces could not bias the movement of tracer to either the dorsal or the ventral region of the eye. Immediately after the start of the injection, fluorescence was found throughout the anterior segment (Fig. 2E) . Within 15 minutes after delivery, most dextran was found ventrally and in association with the ventral canalicular network (Fig. 2F) . Evidence of rhodamine-dextran in the vasculature throughout the fish was also apparent. At 15 minutes the eyes were enucleated, and fluorescence was found in the ventral choroidal rete at the back of the eye (Figs. 2G 2H 2I) . Subsets of these eyes were fixed and further processed for histologic sections. Endothelial cells of the ventral canalicular network showed strong fluorescence (Figs. 2J 2K) . No fluorescence was found in the annular ligament. In addition, little fluorescence was found within the aqueous plexus, suggesting that once the dextran was delivered to this sinus, it was rapidly swept away by blood flow. 

In a final set of experiments, we injected additional tracers into the anterior segment that could be coupled with TEM to better inspect the cellular fate and movement of aqueous humor. We gently injected 10 nm gold-BSA into the anterior chamber over 5 minutes. In the first experiments, the eyes of anesthetized zebrafish were removed and fixed for TEM 10 minutes after the start of the 5-minute anterior segment injection. Ultrastructural analysis revealed the presence of 10 nm-gold-BSA throughout the ventral canalicular network but not within the annular ligament (Fig. 3) . The electron-dense tracer was also found to be endocytosed by ventral canalicular endothelial cells (Figs. 2B 2C) . A small amount of 10 nm gold-BSA was also found in the angular aqueous plexus in association with inner wall endothelial cells (Figs. 3E 3F 3G) . In subsequent experiments, we similarly injected purified HRP and fixed eyes immediately after the 5-minute injection. Eyes were then processed for HRP reactivity and TEM. Consistent with our previous results, we found HRP associated with the surface of the annular ligament, but it had not entered the tissue (Fig. 4B) . HRP reactivity was also associated with the posterior iris epithelium but was not found in the cells or in the iris stroma (Fig. 4C) . HRP reactivity was found in the angular aqueous plexus in what appeared to be a gradient, with the highest concentrations closest to the anterior chamber (Fig. 4D) . Surprisingly, we also found HRP reactivity in association with endothelial cells of ventral vitreal-retinal vessels and also within the vessel lumens (Fig. 4E) . These vessels line the interface of the vitreal-retinal boundary and show a higher density in the ventral portion of the eye. ²⁴ No HRP reactivity, however, was found in the dorsal vitreal-retinal vessels. 

Discussion

With the use of light and electron microscopy with molecular tracers of aqueous humor movement, we have described the aqueous humor drainage pathway of zebrafish. Our data support a model in which aqueous humor is produced in the dorsal hemisphere by the ciliary epithelium and removed through a ventral canalicular network that connects to the angular aqueous plexus (Fig. 5) . This plexus is continuous with the choroidal venous system in which aqueous humor returns to the vascular circulation. Aqueous humor also appears to be taken up by ventrally localized vitreal-retinal vessels. These observations are consistent with those reported for salmonid fish. ³⁴ We did not find evidence of uveal-scleral outflow, though our techniques and timing of tracer experiments were not designed to evaluate this potential secondary route of aqueous outflow. 

Despite the unique vectorial flow of aqueous humor of zebrafish, the local anatomic features and cellular ultrastructure of this system have similarities with the aqueous humor pathway of mammals. Consistent with this observation, average intraocular pressure ranges are conserved between zebrafish and mammals. ³⁵ Anatomic similarities include the morphology of juxtacanalicular tissue overlying the outflow pathway and the morphology and organization of the endothelial cells that line the canalicular network. In particular, endothelial cells show a high degree of junctional coupling and complex cell shapes and intracellular interactions. Canalicular endothelial cells of zebrafish are also highly phagocytic, as are the endothelial cells associated with Schlemm’s canal in mammals. ^{9 36} In addition, a subset of these cells displayed distended or parachuting morphologies. The distended morphologies, resembling giant vacuoles, were most frequent in cells lining the entrance and exit of the canalicular network proximal to the anterior chamber and ventral angular aqueous plexus, respectively. A noted difference with mammals is an absence of obvious trabecular beams. Although other investigators have suggested that the annular ligament of zebrafish represents the trabecular meshwork equivalent, ²⁶ we view this structure as distinct and unique. Instead we suggest the cells overlying the ventral canalicular network at the iridocorneal and ciliary openings are homologous to the juxtacanalicular connective tissue cells of the mammalian trabecular meshwork, and, by extension, the canalicular network is homologous to Schlemm’s canal. 

Like anatomic and tracer experiments in mammals, our studies have not addressed the molecular and cellular mechanisms that facilitate and regulate aqueous humor outflow. In mammals, paracellular and transcellular routes of fluid movement through angle endothelial cells have been proposed. ³⁶ Results from the anatomy and tracer experiments from zebrafish are also consistent with either or both mechanisms. Our anatomic studies also suggest that, as in mammals, the point of major resistance of aqueous humor movement is located at or near the first obvious barrier—the endothelium of the canalicular outflow pathway. Importantly, the description of homologous aqueous humor outflow tissues in zebrafish provides the opportunity for genetic studies to probe the specific molecular and cellular mechanisms that control aqueous humor dynamics and intraocular pressure. 

²  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.

 Supported by Howard Hughes Medical Institute (SWMJ), Pilot grant from The Glaucoma Foundation (BAL), National Institutes of Health Grant R01EY16060 (BAL), and National Eye Institute Core Facilities Grant P30EY001931 to the vision research community of Medical College of Wisconsin.

 Submitted for publication October 15, 2008; revised November 16, 2008; accepted Januray 29, 2009.

 Disclosure: M.P. Gray, None; R.S. Smith, None; K.A. Soules, None; S.W.M. John, None; B.A. Link, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Anatomy of the iridocorneal angles of adult zebrafish. (A) Schematic of tissues in the adult zebrafish eye. In all panels dorsal is up, ventral is down, anterior is left, and posterior is right. (B) Toluidine blue–stained semithin section montage of a dorsal iridocorneal angle. (B′, inset) High-magnification TEM of pigmented and nonpigmented epithelia of the dorsal ciliary region. (C) Semithin section montage of a ventral iridocorneal angle. (D) Higher magnification of endothelial canalicular tissue and adjacent structures, indicating area for TEM analysis (red boxed regions). Black arrow: iridocorneal canal opening; white arrow: ciliary canal opening. (E–I) High-magnification TEM (insets) of cells in the ventral canalicular network. Note the (G, white arrowheads) numerous coated pits and micropinocytic vesicles and the (H, I) tight and adherens junctions between endothelial cells. (J–M) TEM of canalicular endothelia and openings. Note the (J) loosely organized juxtacanalicular connective tissue (JCT) at the opening of the canaliculi and the (M, white arrowheads) parachuting morphology and giant vacuoles of endothelial cells adjacent to the angular aqueous plexus. Scale bars, 1.0 μm (B′, K, L); 0.5 μm (M). VRVs, vitreal-retinal vessels (red); NR, neural retina (pink); RPE, retinal pigment epithelium (black); ON, optic nerve (pink); sclera (gray); SO, scleral ossicle (dark blue); AAP, angular aqueous plexus (red); endothelial canaliculi (green); cornea and lens (light blue); annular ligament (purple).

Figure 5.

 

Model of aqueous humor dynamics in the zebrafish eye. (A) Overview showing the vectorial flow of aqueous humor (blue arrows) from the dorsal ciliary epithelium to the ventral canalicular network and ventral vitreal-retinal vessels. (B) Higher magnification of aqueous humor outflow indicating absorption into the iridocorneal and ciliary openings of the ventral canalicular network and the ventral vitreal-retinal vessels. (C) Higher detail of the outflow pathway showing juxtacanalicular connective tissue cells at the iridocorneal and ciliary openings (indicated by arrows) and the tortuous lacunae created by endothelial cells (green) lining the canalicular network; for comparison see Figure 1D . Lens and cornea (light blue); AL, annular ligament (purple); blood-filled vessels and sinuses (red); iris argentea (yellow); iris stroma, lentis retractor, and sclera (gray); NR, neural retina (pink); scleral ossicle (dark blue); aqueous humor in outflow tissues (blue-white dots in A and B; pale blue in C).

Figure 2.

 

Tissue fate of aqueous humor tracers in zebrafish eyes. (A, B) Peroxidase staining to localize secretion and absorption of 3 kDa biotin-dextrans injected into the ophthalmic artery. Eyes were enucleated and fixed after (A) 2 minutes or (B) 10 minutes. Note the dorsally localized peroxidase product (A, white arrowheads) and the ventrally localized staining in A and particularly B (white asterisks). (C) Bright-field microscopy of anesthetized zebrafish eye before anterior segment injection of 3 kDa rhodamine-dextran. (D–F) Fluorescence microscopy of anesthetized zebrafish eye (D) during, (E) 5 minutes after, and (F) 15 minutes after anterior segment injection of 3 kDa rhodamine-dextran. (G–I) Ventral view of isolated eyes after anterior segment injection of 3 kDa rhodamine-dextran showing bright-field microscopy (G), fluorescence microscopy (H), and overlaid images (I). Note the concentrated fluorescence in the ventral iridocorneal angle and choroidal tissues (white arrows). (J) Fluorescence microscopy of semithin section of ventral endothelial canaliculi after anterior segment injection of 3 kDa rhodamine-dextran. (K) Bright-field microscopy of section equivalent to the previous section (J) stained with toluidine blue to highlight the relative locations of the endothelial canaliculi, angular aqueous plexus, scleral ossicle, and neural retina.

Figure 3.

 

Movement of 10 nm gold-BSA through the ventral canalicular network. (A) Schematic of the ventral angle to indicate locations of TEM images. After injection of 10 nm gold-BSA into the anterior chamber, zebrafish eyes were fixed and processed for TEM. (B–D) TEM showing electron-dense 10 nm gold-BSA localized to coated endocytic pits of canalicular endothelial cells (C) and within open spaces between endothelial cells (D). (E–G) TEM of endothelial cells lining the ventral angular aqueous plexus and highlighting 10 nm gold-BSA at the termination of the outflow pathway.

Figure 4.

 

Peroxidase activity after HRP injection in the anterior chamber. (A) Schematic of the ventral angle to indicate locations of TEM images. (B) Dark-appearing peroxidase activity is associated with the epithelium covering the annular ligament (arrowheads) but is not found within or between annular ligament cells (asterisks). (C) Similarly, peroxidase activity is associated with the nonpigmented epithelial surface of the iris (arrowheads) but not within or between epithelial or stromal cells (asterisks). (D) A sinus of the angular aqueous plexus showing peroxidase activity (fluid surrounding the RBCs, arrows). (E) Peroxidase staining associated with endothelial cells (arrowheads) and within the lumen of vitreal-retinal vessels (fluid surrounding the RBC). No staining was noted in the nerve fiber layer of the neural retina (asterisks). RBC, red blood cell.

The authors thank Olga Savinova and Michael Cliff for assistance with sectioning and animal husbandry, Clive Wells and Lesley Bechtold for assistance with TEM, and Xinping Zhao for helpful discussions and sharing unpublished data that confirmed some of our studies.