The choroidal vascular system has been a recent focus of interest in attempts to interpret pathologic features of the choroid. The understanding of pathologic processes in the choroid requires an exact knowledge of the details of normal anatomy.
The choroidal vasculature has been extensively investigated in humans (Weiter and Ernest, 1974; Woodlief and Eifrig, 1982; Yoneya and Tso, 1987; Olver, 1990; McLeod and Lutty, 1994) as well as in experimental animals such as monkeys (Shimizu and Ujiie, 1978; Risco et al., 1981), rabbits (Funk and Rohen, 1987), guinea pigs (Fukuda and Matsusaka, 1977), cats (Wong and Macri, 1964), dogs (Van Buskirk, 1979), cows (Kohler and Leiser, 1983), and newts (Lazzari et al., 1993) by various methods, including histology, India ink injections, flat preparations, neoprene latex and plastic vascular casts, and alkaline phosphatase staining.
Although the general distribution of the choroidal vasculature of primates and other species has been described, it is surprising that adequate descriptions of the choroidal vasculature of small animals such as rats and mice have been few. Small animals are considered to be useful experimental models for several uveal diseases, because it is easy to treat small animals. There have been many studies on the microvasculature of the rat retina (Pannarale et al., 1991; Bhutto and Amemiya, 1995a,b, 1997) and optic nerve head (Morrison et al., 1999; Sugiyama et al., 1999), but only a few have dealt with the microvascular architecture of the rat choroid by using the corrosion cast method with a scanning electron microscope (Matsusaka, 1976; Yoshimoto et al., 1980). These studies have focused on the peripapillary region and the posterior choroid. Therefore, we need to demonstrate the normal morphology throughout the rat choroid before pathologic experiments. Nonhuman primates are anatomically the most appropriate animals for the study of human disease (Anderson, 1969). However, the expense and limited supply restrict their use in careful experiments requiring a large number of animals.
The production of vascular corrosion casts suitable for scanning electron microscopy has opened a new perspective for the study of the vascular morphology of the eye in vitro. This technique offers unique three-dimensional pictures of the origin, size, course, branching pattern, and spatial arrangement of the blood vessels at high resolution and with great depth of focus.
With the success of our corrosion cast studies on the rat retinal vasculature (Bhutto and Amemiya, 1995a,b), we extended this technique to the observation of the rat choroid. We first tried to establish the normal anatomy of the rat choroid, i.e., details of the three-dimensional microvascular anatomy of the rat choroid by using corrosion casts and scanning electron microscopy.
MATERIALS AND METHODS
Twenty male and female Wistar Kyoto rats, 12–24 weeks after birth, weighing 250 to 350 g with a normal ocular fundus, were fed a standard laboratory chow diet (Oriental Kobo) and given tap water ad libitum. All rats were kept in a room under controlled temperature (21 ± 2) and humidity (55 ± 5%) with a 12:12 hr light/dark cycle (light period, 07:00–19:00) in the Laboratory Animal Center for Biomedical Research, Nagasaki University School of Medicine. The experiment was carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Corrosion Casts and Scanning Electron Microscopy
Rats were anesthetized with intraperitoneal sodium pentobarbital (25 mg/kg body weight). The vascular casts were prepared by our previously described technique (Bhutto and Amemiya, 1995a). In brief, both common carotid arteries were cannulated and the jugular veins were then cut. The vascular system was perfused with heparinized normal saline solution (500 IU/100 ml), and freshly prepared Mercox CL-2B resin (Dainippon Ink and Chemicals, Inc., Japan) was injected into the cannulated carotid arteries. Then the eyeballs were enucleated and left in a warm water bath (56°C) for a few hours to allow polymerization and tempering of the resin. When the polymerization was completed, the ocular tissues were macerated for 5–6 days by repeated baths in 20% potassium hydroxide at room temperature followed by washing with running tap water. Microdissection was done with fine tweezers and scissors under a binocular light microscope to expose the choroidal vasculature. Casts were again gently washed with running tap water and digested a second time for 1–2 days to remove residual tissues. The casts were desiccated by freeze-drying, and the dried vascular casts were impregnated with osmium overnight, then mounted on scanning electron microscope (SEM) stubs with double-sided adhesive tape and coated with ion spatter gold palladium. The casts were examined with a Hitachi S-2360N SEM at an accelerating voltage of 6 to 10 kV.
The choroid casts were viewed from both anterior and posterior aspects of the globe. At first, we observed the anterior (scleral side) aspect of choroid casts. Then, the mounted casts were removed with a razor (Feather-S) blade and remounted to determine the posterior (retinal side) aspect. After remounting, the casts were recoated with gold-palladium to obtain electron microscopic scans of the choriocapillaris. The caliber of the choroidal vessels and the choriocapillaris was measured with a caliber micrometer in photographs enlarged two times the original magnification.
The corrosion casts provided excellent three-dimensional visualization of the entire choroidal vasculature of the rat (Fig. 1). All casts demonstrated complete filling of the major arteries and veins of the choroid with extensive filling of the fine capillary bed in a majority of specimens.
The main arterial supply to the rat eye came from the posterior ciliary artery (PCA) derived from the inferior branch of the ophthalmic artery. This vessel entered the eye from the inferior-nasal quadrant of the optic nerve. In this course, the PCA sent off branches to the distal part of the optic nerve, forming a dense vascular annulus. From this ring plexus emerged branches for the retina and choroid, which pierced the sclera adjacent to the optic nerve.
Immediately posterior to the globe as revealed by corrosion casts, the PCA trifurcated into the central retinal artery and nasal (diameter, 126.41 ± 16.81 μm; n = 20) and temporal (diameter, 124.62 ± 14.03 μm; n = 20) long posterior ciliary arteries (Fig. 2). The main stems of the nasal and temporal long posterior ciliary arteries (LPCAs), which supplied the entire choroid passed toward the anterior segment of the eye, forming the iridociliary circle. The temporal long posterior ciliary artery usually sent off an inferior branch (110.52 ± 12.3 μm in diameter, n = 10) to the lower half of the choroid (Fig. 2). A comparable artery for the upper half did not exist, but instead, the nasal and temporal long posterior ciliary arteries formed branches supplying the upper half of the choroid. The choroidal arteries and veins between the optic disc and the half of the posterior choroid were arranged in an interlacing pattern (Fig. 3). In the posterior choroid, the LPCAs formed five to seven branches on each side, supplying the adjacent choriocapillaries before they reached the equator. These arterial vessels repeatedly branched off in the same plane and ran in a relatively straight course and made a right-angle turn forward, and were assumed to become smaller before supplying to the choriocapillaris (Fig. 4). The vessel diameters were 126.41 ± 16.81 μm (n = 20) in the LPCA, 67.83 ± 9.06 μm (n = 16) at the root of the first branch from the LPCA, and 38.54 ± 6.70 μm (n = 16) at the root of the first sub-branch from the first branch. These precapillary arterioles were arranged regularly. In contrast, the inferior branch (IF) artery showed a dichotic ramification (Fig. 5), beginning shortly after it branched from the main LPCA, and precapillary arteriolar branches ran in the same manner as precapillary arterioles in the other regions. Arterio-arterial anastomoses between the main branches of the ciliary arteries were not observed. In each quadrant, from the center to the periphery, the choroidal vessels on the surface were densely arranged. Because of the density of these vessels, it was difficult to trace the arterioles into the choriocapillaris. Thus, we could not confirm how terminal arterioles entered the choriocapillaris.
Anterior to the equator, the LPCAs divided once and sent off branches that formed the circular ciliary artery. Slightly distal to the origin of the circular ciliary artery, branches of LPCAs emerged to form the circumlimbal artery (Fig. 6). This artery ran superficially in the sclera and gave rise to anastomosing arches located in a 2–3 mm zone behind the limbus. The arches sent off fine radial twigs to the corneal region, which represented the arterial components of the perilimbal vascular network.
Finally, the LPCAs entered the iris at its root and bifurcated symmetrically to form the circular iris artery (Fig. 7); it lies midway between the insertion of the iris and the pupillary margin. Many fine branches of the iris artery proceeded to the pupillary margin (radial iris arteries) or posteriorly to the ciliary body (radial ciliary arteries). The radial iris arteries were arranged in a simple pattern almost without cross-connections. The radial ciliary branches underwent further subdivisions as they traveled toward the base of the iris. The branches arising at this site were distributed to the vascular bed in the ciliary muscle and played a role for the main arterial supply to the ciliary processes. The anterior ciliary arteries, per se, were absent in the rat.
The choriocapillaris, viewed from the retinal side, appeared as a nonhomogeneous network of capillaries with different diameters. This monolayer vascular network consisted of a dense honeycomb pattern of capillaries with diameters of 15.25 ± 1.97 μm (n = 36), and an irregular pattern of capillaries with diameters of 14.18 ± 2.21 μm (n = 36) (Fig. 8). These two patterns in the choriocapillaris were distributed throughout the vascular surface of the choroid except in the peripheral area. Where the choriocapillaris, which was mostly venous in nature, formed a more elongated palm-like vascular pattern of arcades and finally terminated at the ora serrata (Fig. 8).
There was no evidence of a lobular arrangement of the choriocapillaris. At higher magnification, in the peripapillary area, the irregular pattern was evident (Fig. 9a). In the posterior pole, the honeycomb pattern appeared as a dense network of capillaries (Fig. 9b), fairly uniform in caliber and branching frequently, which interconnected freely to form a network with smaller intercapillary meshes. The irregular pattern of intercapillary meshes was wider and longer than the honeycomb pattern (Fig. 9c). In the peripheral area, the irregular pattern was more open and wider (Fig. 9d), the branching was less frequent, and the caliber of the capillaries was more variable (12.8 ± 2.08 μm in diameter, n = 36) than in the posterior pole and peripapillary area. In addition, in the peripheral area of the choriocapillaris, horizontal lines of vascular dilatations between capillaries were sometimes observed (Fig. 8). In many cases, no larger vessel was detected behind these vascular structures. This kind of vascular network enables one to trace the distribution of the arteriolar and venular branches, capillaries, and their connections.
Two distinct venous outflow patterns of the rat choroid were observed (Fig. 1). (1) The venous blood from the central region, peripapillary choroid, and sometime optic nerve head was drained by smaller veins, which ran more or less directly into the posterior ciliary vein. (2) The venous blood from the posterior half of the choroid, anterior choroid, and veins from the iris, ciliary processes, and some small veins from the corneo-scleral region drained into the vortex veins.
The small veins around the papillary region and the veins running parallel bilaterally along the long posterior ciliary arteries from nasal and temporal sides joined around the optic nerve head to form venous sinuses that drained into the peripapillary choroidal veins, which probably connected directly with posterior ciliary vein (Fig. 10). At the posterior choroid, from half of the course between the arterioles, dense collecting venules with various diameters, which filled up spaces among arterial branches, converged into the vortex veins. The choroidal veins ran parallel to the arterioles and were tortuous and branched even more frequently than did the arteries (Fig. 4). The diameters of the veins and venules were larger than those of the arteries and arterioles. Venous collateral channels and intervenular anastomoses were frequently observed between collecting venules, and venules to larger veins converged to form ampullae. Collecting veins from the anterior choroid also drained toward the vortex vein. The larger veins, six or seven in number, converging from all directions toward the vortex vein usually formed two ampullae, sometimes three, in each quadrant (Fig. 11). The diameter of an ampulla was 298.83 ± 36.74 μm (n = 18). These ampullae drained blood into the four individual vortex veins located on the dorsal, ventral, nasal, and temporal sides, which pierced the sclera just posterior to the equator of the eyeball. The main vortex vein, which passed from the ampulla out through the sclera, was slightly narrower.
The vortex veins also drained most of the venous blood from the iris and ciliary body (Fig. 12). The veins draining the blood from the iris ran between the ciliary processes and generally drained the blood into the choroidal veins. Some of the large iris veins drained the blood directly into the ampullae of the vortex vein. In addition, some small veins from the corneoscleral region drained blood into the choroidal veins. The above-mentioned findings were common in rats aged 12–24 weeks.
The rat choroidal vasculature has been studied with the corrosion cast method, but, to the best of our knowledge, this is the first detailed description of the arteries, choriocapillaris, and venous drainage of the choroid in rats. We could observe important similarities and differences between rat and primate choroids.
The rat choroidal vasculature is unique and simpler than that of primates and many other animals. In primate eyes, the short posterior ciliary arteries and branches of the ophthalmic artery form a circle around the optic nerve as they pass almost perpendicularly through the sclera to supply the choroid (Sugiyama et al., 1994). In rabbits, medial and lateral posterior ciliary arteries arise from the ophthalmic artery and are at some distance from the optic nerve; after sending branches to the optic nerve, they supply most of the choroid (Sugiyama et al., 1992). In rats, however, the posterior ciliary artery derived from the inferior branch of the ophthalmic artery (Greene, 1963) travels through the inferior side of the optic nerve sheath toward the optic nerve head, sends off branches to the optic nerve (Sugiyama et al., 1999), and becomes the main arterial supply to the entire uvea. The present study supports earlier observations on rat uvea that the posterior ciliary artery gives off a central retinal artery and two long posterior ciliary arteries in the optic nerve head region, provides several branches to the choroidal vasculature and finally supplies the iris and ciliary body vasculature (Yoshimoto et al., 1980; Funk and Rohen, 1985; Funk, 1986; Morrison et al., 1995). That we did not find the circle of Zinn-Haller in rats' eyes supports the findings of Sugiyama et al. (1999) and suggests that the rat's posterior ciliary artery is a terminal artery in the eyeball and that optic nerve axotomy inevitably cuts the posterior ciliary artery and produces ischemia in the overall ocular vasculature.
The present study showed an interesting aspect of the venous system. The principal vein in the choroidal circulatory system has long been known to be the vortex vein (Duke-Elder and Wybar, 1961; Hayreh, 1964), and the existence and nature of other veins have been largely ignored (Duke-Elder and Wybar, 1961; Hogan et al., 1971). It has been noted that in the rat, as well as in other species, four well-developed vortex veins are present, but the distribution of these veins to the posterior half of the choroid has not been reported. We found that the venous blood from the central region, peripapillary choroid, and sometime optic nerve head was drained by smaller veins, which might run directly into the posterior ciliary vein. We assume that they are the primary drainage pathway of the posterior choroid in the rat and that they provide evidence of the existence of a new drainage pathway of the choroidal circulatory system, which does not travel by means of the vortex vein.
Interarterial, intervenous, and arteriovenous anastomoses are well-known morphologic features of the choroidal vascular architecture (Hayreh, 1975; Shimizu and Ujiie, 1978; Yoneya and Tso, 1987). These vascular connections are randomly distributed in the choroidal medium-sized and large vessel layers and are thought to be important in the complicated choroidal blood circulation as it responds to regional choroidal needs under both physiologic and pathologic conditions. Matsusaka (1976) described medium-sized arteries and veins forming interarterial and intervenous shunts in the rat choroid; this finding is similar to that described in monkeys (Shimizu and Ujiie, 1978). We did not find interarterial or arterio-venular shunts. However, intervenous channels and anastomoses were frequently noted. The function of the venous channels and anastomoses cannot be demonstrated by morphologic studies, but it may be speculated that these help blood flow in the choroid.
Anatomically, the choriocapillaris is not a homogeneous structure but varies from the peripapillary to the peripheral areas, and the choroid is not entirely “lobular.” Our anatomic study of the rat choroid supports previously described observations (Ashton, 1952; Klien, 1966; Weiter and Ernest, 1974; Krey, 1975; Shimizu and Ujiie, 1978; Woodlief and Eifrig, 1982; Yoneya and Tso, 1987) that the appearance of the choriocapillaris is not homogeneous throughout but varies from the peripapillary to the peripheral areas. In the present study, vascular casts viewed from the retinal aspect showed that the peripapillary as well as the posterior pole capillaris have a dense network of freely connected capillaries that resemble a honeycomb, giving the impression that an extensive pool of blood is formed by numerous channels composed of intervascular matrix. This pattern might change to a palm-like pattern. The choriocapillaris ends in the ora serrata forming arcades. In the peripheral area, the choriocapillaris meshwork is wider than in the posterior pole. In addition, in the peripheral area of the choriocapillaris, horizontal lines of vascular dilatations between capillaries were observed. In many cases, no larger vessel was detected behind these vascular structures. Their role in the choroidal microcirculation is not yet known. We speculate that they may act as a regulatory valve between the anterior and posterior parts of the choroidal circulation in respect to the equator. However, this mechanism must be determined by functional studies.
Our study of the rat choroid did not show a lobular arrangement of the choriocapillaris. Boundaries of lobules are sometimes poorly defined. Choroidal lobules may be round, oval, octagonal, irregular, or a mosaic pattern composed of three- to six-sided vascular units (Torczynski and Tso, 1976) consisting of a meshwork of capillaries in a radial and circumferential arrangement. In the present study, because of the density of the vessels on the choroidal surface, it was difficult to trace the arterioles and venules. Even when the casts were viewed from the retinal side to determine the location of a feeding arteriole or a draining venule due to the prevailing of a dense honeycomb pattern of the choriocapillaris, we were unable to determine how these vessels entered or exited. We could not even break the casts to trace the course of these vessels. Generally speaking, vascular casts of a rat choroid are very tiny, weak, and fragile. Even rinsing the vascular casts with water and freeze-drying them could destroy details of complete microvascular casts, although such a destruction of vascular casts never occurred in the present study.
No fenestration of the choriocapillaris was seen. When the choriocapillaris was viewed from the retinal side at higher magnification, the vascular cast surface exhibited some protrusion-like imprints of various sizes and shapes, which probably represented endothelial fenestrations of the choriocapillaris, but this appearance was not always observed in all specimens. It might have represented extravasations of resin through fenestration gaps between endothelial cells. We could not avoid the possibility of artifacts in corrosion cast preparation, although we succeeded in making almost complete choroidal vascular casts.
In the present study, we also measured the caliber of choroidal vessels: 126.41 ± 16.81 μm in the LPCA, 67.83 ± 9.06 μm at the root of the first branch from the LPCA, and 38.54 ± 6.70 μm at the root of the first sub-branch from the first branch. Thus, the ratio of these vessels' diameters is 4:2:1. The injection of resin without fixation may result in expanding of the vessel diameter to two or more times the physiological diameter. At the beginning of the present study, the authors tried to make vascular corrosion casts with fixed materials but found that it was very difficult to make good specimens with fixed retina and choroid but was relatively easy with unfixed materials. The use of perfusion fixation before casting (prefixation) may induce vascular constriction and also diminish the elasticity of the vessels, hindering sufficient injection of the casting medium into the capillary bed (Ohtani and Murakami, 1992). In addition, the manual injection of viscous methyl methacrylate may produce pressures considerably different from normal in the choroid, so the diameters of the blood vessels may not be the same as in vivo diameters. Even so, the diameters and densities of the choroidal vessels are useful as standard data in evaluating the changes seen in pathologic conditions of the uvea, if we compare the data obtained under the same experimental conditions.
Our study shows that the angioarchitecture of the rat choroid varies distinctly in different locations, supporting observations that marked regional variations in humans and other primates reflect differences in choroidal blood flow. Resistance to blood flow is a common factor in various structures in the arterial system and in the choriocapillaris. In the venous system, the exclusion of resistance is the main object. In the present study, a venous structure suggests such a “down-hill” phenomenon. For example, in venous casts one can see a gentle slope forming a loop and marked dilation of a vein at its connection with capillaries (Fig. 11). Such a change in caliber was also noted at the entrance into a vortex vein.
With an accurate picture of the angioarchitecture of the rat choroid, we are now able to demonstrate various specific patterns in choroidal diseases, and this anatomic knowledge will help us to better understand choroidal pathology. The distinct vascular patterns between the posterior pole and the peripheral areas may be changed by chorioretinal diseases. Our observations indicate that the rat choroidal vasculature differs from that of humans and other primates. Despite these interspecies differences, the establishment of a thorough baseline concept of choroidal vasculature in a readily accessible experimental model may be a useful addition in studies of choroidal pathology.