Limbal Microvasculature of the Rat Eye

John C. Morrison*^ F. W. Fraunfelder,* Scott T. Milne* and Chester G. Moore* f

Purpose. To define the microvascular anatomy of anterior segment blood supply and aqueous drainage in the rat eye. Methods. External limbal blood vessels were studied by direct inspection of normal, living rat eyes and eyes injected intracamerally with fluorescein dye. Intraocular connections of these vessels were then documented with scanning electron microscopy of methylmethacrylate microvascular luminal castings. Results. The rat limbus possesses a circumferential vascular ring consisting of a single artery and a venous plexus. The limbal artery communicates with radial anterior ciliary arteries and with perforating arterioles arising from the long posterior ciliary arteries. The venous plexus is connected to a circumferential Schlemm's canal by numerous transcleral, aqueous-containing collector channels and drains into multiple radial veins located within die episclera. Conclusions. These findings illustrate anatomic similarities between rats and primates, both in anterior segment blood supply and aqueous humor drainage. The rat limbal artery provides collateral perfusion of the anterior segment from anterior and long posterior ciliary systems. The direct communications between identifiable external aqueous-containing veins, a circumferential episcleral venous plexus, and an internal Schlemm's canal provides the anatomic basis for producing chronically elevated intraocular pressure in rats using retrograde injection of mild sclerosing agents into the aqueous humor outflow pathways, and for administering drugs and other agents to the entire trabecular meshwork and Schlemm's canal. Invest Ophthalmol Vis Sci. 1995;36:751-756.

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Continued progress in understanding glaucomatous optic nerve damage relies on the development of appropriate and cost-effective animal models. Although analyses of enucleated human glaucomatous eyes have provided much information,1'2 studies in live monkeys with experimentally elevated intraocular pressure (IOP) were required to corroborate these findings and to test the resultant theories of damage.3'4 Spontaneous glaucoma has been described in numerous animals,5"10 and many animal models of exFrom the * Kenneth C. Swan Ocular Neiirobiology iMboratory, Casey Eye Institute, and the ^Portland Veterans Administration Hospital and Medical Center, Portland, Presented in part at the 1992 annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida. Supported by grants from the Medical Research Foundation of Oregon, The Veteran's Administration (1-2-409-540), and the National Institutes of Health (EY10145-01 toJCM and RR00163 to the Oregon Regional Primate Research Center); by Sigma Xi's Grants-in-Aid of Research; and by Research to Prevent Blindness. JCM is a Research to Prevent Blindness Miriam and Benedict Wolfe Scholar. Submitted for publication June 9, 1994; revised September 14, 1994; accepted October 6, 1994. Proprietary interest category: N. Reprint requests: John C. Morrison, Casey Eye Institute, Oregon Health Sciences University, 3375 S.W. TerwiUigerBoulevard, Portland, OR 97201-4197.

perimental glaucoma have been described, including elevation of IOP by specific treatment of the trabecular meshwork with laser9"12 or by particle injections. 91013 Most of these models involve closure of the anterior chamber angle and are suitable primarily for studying the effects of elevated IOP on the optic nerve. Nonspecific treatments using steroids,14"18 water loading,19'20 or continuous light exposure21 have also been described, and although trabecular damage is minimal, the IOP elevation is usually bilateral. Some of these models are anatomically inappropriate, whereas others are unavailable. Our previous demonstration that IOP in rats can be measured noninvasively with a tonopen 22 has recently received independent confirmation.23 This makes this common laboratory animal attractive as a potential model for glaucoma research. We used direct inspection of living eyes and scanning electron microscopy of microvascular castings from laboratory rats to determine the microvascular anatomy of the rat limbus. The results provide a basis for understanding anterior segment blood supply and aqueous humor

Investigative Ophthalmology & Visual Science, March 1995, Vol. 36, No. 3 Copyright Association for Research in Vision and Ophthalmology

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FIGURE l. In vivo photograph of rat limbus showing anterior ciliary artery (asterisk) and aqueous-containing veins (arrows) supplying limbal artery and venous plexus, respectively. Original magnification, X60.

FIGURE 2. Rat limbus after injection of fluorescein dye into the anterior chamber. Unfilled limbal artery stands out against the dye-filled limbal venous plexus (arrowheads). Dyefilled, radial aqueous-containing veins are also visible (arrows). Original magnification, X15.

drainage in the rat eye, two major determinants of aqueous humor dynamics and intraocular pressure. MATERIALS AND METHODS All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty adult brown Norway and albino Lewis rats were anesthetized by intraperitoneal injection of a 1.5 ml/kg solution containing 5 ml ketamine (100 mg/ml), 2.5 ml xylazine (20 mg/ml), 1 ml acepromazine (10 mg/ml), and 1.5 ml sterile water. All animals were examined with a Wild M3Z dissecting microscope (Wild Leitz; Heerbrugg, Switzerland) to assess general features of the external limbal vasculature. The anterior chambers of four animals were injected with a 1:20 dilution of fluorescein dye using a 32-gauge needle. The pattern of dye drainage from the anterior chamber was then documented using a slit lamp camera equipped with a medium SE-40 blue excitor filter. For microcorrosion casting studies, methylmethacrylate casting medium was prepared by diluting Batson's #17 base (Polysciences, Warrington, PA) with an equal volume of methylmethacrylate monomer (Aidrich, Milwaukee, Wl). This mixture was divided into two separate containers, adding catalyst to one and promoter to the other as previously described.24 The abdominal aortas of 16 rats were isolated and cannulated retrogradely with 20-gauge catheters secured with 5-0 silk sutures. The separate catalyst and promoter mixtures were then combined, mixed for 30 seconds, and aspirated into plastic 60-ml syringes. Casting medium was injected by moderate thumb

pressure into the abdominal aorta, and the right auricle was punctured to allow efflux of blood and plastic from the head. The corneas were also incised before injection of casting medium to reduce intraocular pressure and maximize filling of intraocular blood vessels. Incisions did not exceed 2.5 mm. Injection was continued until a noticeable increase in viscosity occurred. After allowing 1 hour for polymerization, the eyes were enucleated and placed in 4% paraformaldehyde for 24 hours. The tissue was then digested in 6 M KOH solution overnight at 60C, and the resultant castings were gently washed with distilled water and allowed to air dry. Castings were then mounted, coated with gold palladium (40 nm), and examined under an AMR 1000 scanning electron microscope (Amray; Bedford, MA). Scanning electron microscopy and sequential microdissection2' were employed to document the details of the limbal blood vessels and their intraocular connections. Casts of arteries and veins were identified by their distinctive endothelial nuclear imprint pattern, the former appearing more fusiform and linear. RESULTS Direct observations revealed a perilimbal vascular ring composed of a single artery and a venous plexus in all animals (Fig. 1). The limbal artery was supplied externally by anterior ciliary arteries located in the episclera. The venous plexus was drained posteriorly by several radial veins within the episclera and conjunctiva. Fluorescein dye injected into the anterior chamber escaped

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FIGURE 3. External view of microvascular casting demonstrating limbal vascular ring composed of single artery (arrow) and venous plexus (arrowhead), the latter connecting to aqueous-containing v i i s (AV). Asterisk indicates capillary bed, which is connected to the limbal venous plexus. Casting orientation conforms to orientation of Figures 1 and 2. Original magnification, X90. rapidly into the limbal and episcleral veins, illustrating the importance of these vessels as a route for aqueous humor outflow (Fig. 2). The limbal artery did not fill with fluorescein and appeared in silhouette against the dye-filled limbal veins. Scanning electron microscopy of methylmethacrylate luminal castings confirmed the presence of a sin-

gle limbal artery with the venous plexus (Fig. 3). Careful microdissection revealed intraocular connections of the limbal artery to the long posterior ciliary arteries in the form of limbal perforating arterioles (Fig. 4). These perforators, two per eye, were consistently found nasally and temporally, just before bifurcation of the long posterior ciliary arteries into the major arterial circle. Externally, the multiple channels of the limbal venous plexus were found adjacent to the limbal artery (Fig. 3). Internally, careful microdissection revealed a cast of Schlemm's canal, which appeared as a flattened channel lying deep to the limbal plexus but external to the iris vessels (Fig. 4). Numerous transcleral collector channels were found connecting Schlemm's canal to the limbal venous plexus (Fig. 5), providing an anatomic pathway for intracameral fluid to gain access to the episcleral limbal veins. All castings without obviousfillingdefects revealed that the venous plexus was a complete ring around the entire limbus (Fig. 6A). The extremely delicate nature of the casting prevented the successful isolation of the Schlemm's canal in its entirety, although circumferentially oriented portions, often several clock hours in length, were frequently noted (Fig. 6B). No major differences from these overall patterns, summarized in Figure 7, were noted among any of die animals and castings studied. No differences in either blood supply or aqueous humor outflow patfiways were seen between pigmented and nonpigmented animals. DISCUSSION Although specific descriptions of rat ciliary process capillary beds and the trabecular meshwork exist, this

FIGURE4. Limbal artery (arrow) connects internally with the long posterior ciliary artery (asterisk) through a perforating arteriole near the origin of the major arterial circle. This partially dissected specimen also shows the relationship of Schlemm's canal (SC) and limbal venous plexus (arrowheads) to limbal and long posterior ciliary arteries. Original magnification, X90.

FIGURE 5. Anterior view of casting demonstrates Schlemm's canal (SC) communicating with the limbal plexus (asterisk) through a transcleral collector channel (arrow). Original magnification, X375.

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is the first overall study of the major blood vessels responsible for serving the anterior segment of the rat eye. We have defined a circumferential limbal vascular plexus that has arterial and venous components and offers the potential for affecting aqueous humor formation and drainage. Although prepared under nonphysiologic conditions, the casts illustrated here do not show significant artifacts offillingor plastic extravasation. As such, they show the potential for blood and aqueous flow, and the anatomic possibilities represented allow speculation on their significance. Although previous work has demonstrated several similarities in the microvasculature of rat and primate ciliary processes/1' our observations indicate that rats

LPCA

AV
ACA

FIGURE 7. Diagram of the rat limbal microvasculature, illustrating the association of arteries (light outline) and veins (heavy outline). Limbal artery (arrows) is supplied by anterior ciliary arteries (ACA) and long posterior ciliary arteries (LPCA), providing collateral blood supply for the iris (I) and ciliary processes (CP). Venous plexus (asterisk) is connected to Schlemm's canal (SC) through multiple collector channels (arrowheads) and drains into radial aqueous-containing veins (AV).

possess further similarities to primates with regard to overall anterior segment blood supply. In rats, the arterial component of the limbal vascular plexus, the limbal artery, is supplied externally by anterior ciliary arteries. In addition, the limbal artery communicates with the intraocular long posterior ciliary arteries just before bifurcation into the major arterial circle. Because arterioles for die iris and ciliary processes arise from the major arterial circle, the rat anterior uvea has the potential, through the limbal artery, to receive collateral blood supply from the anterior and the long posterior ciliary arteries. This is analogous to primates27 and reflects the importance of maintaining adequate ciliary body perfusion under varying physiologic and pathologic conditions.28"32 Conventional light and electron microscopic studies have previously demonstrated that rats possess an identifiable Schlemm's canal and trabecular meshwork with an ultrastructure similar to that of primates.33"35 Our methylmethacrylate castings not only confirm the presence of a Schlemm's canal in rats, they illustrate the overall anatomy of aqueous humor access to the general circulation. Lowering eye pressure before injecting methylmethacrylate, a technique successfully used to demonstrate aqueous humor outflow pathways in other species,36 allowed successful retrograde filling of Schlemm's canal, which appears to be an extensive FIGURE 6. (A) Montage of a single, complete, circumferential and apparently complete channel. The connection of limbal venous plexus with numerous aqueous-containing veins (arrows). Original magnification, xlf>. (B) Magnified Schlemm's canal, through transcleral collector channels, to the episcleral venous plexus and then to radial view of lower right-hand corner of montage in 6A, demonstrating a segment of'Schlemm's canal (arrows), lying inter- episcleral veins confirms that the rat trabecular meshnal and parallel to the limbal plexus (arrowheads). Original work and Schlemm's canal are responsible for at least a portion of aqueous humor drainage, analogous to magnification, X55.

Limbal Microvasculature of the Rat Eye the "conventional" route of aqueous humor outflow in primates.37 The concentric, interconnected Schlemm's canal and limbal venous plexus shown in this study present a double, circular drainage system for aqueous humor into the general circulation. This offers the potential of introducing agents to the entire trabecular meshwork through a retrograde injection into a single aqueous-containing vein. Using a specially designed microneedle, we recently demonstrated the feasibility of such an injection using hypertonic saline.38 This method produces scarring of aqueous humor outflow pathways, resulting in prolonged elevation of IOP and a new potential model of chronic IOP-induced optic nerve damage in rats.
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32. 33. 34. 35.

Announcement
Call for Nominations for the Jin H. Kinoshita Lectureship
The National Foundation for Eye Research, an organization dedicated to the support of cataract research, is pleased to announce a call for nominations for the third Jin H. Kinoshita Lectureship (honorarium, $15,000) to be given at the International Cooperative Cataract Research Group meeting in Kallua-Rona, Hawaii, November 15-19, 1995. Nominations should include a curriculum vitae and a supporting letter. The Nomination Committee consists of Suguru Fukushi, MD; Joseph Horwitz, PhD; Venkat N. Reddy, PhD; and Abraham Spector, PhD, Chair. The deadline for nominations is June 1, 1995. Please send nominations to: Dr. Abraham Spector Department of Ophthalmology College of Physicians & Surgeons of Columbia University 630 West 158th Street New York NY 10032