Exp. Eye Res. (1978) 27, 159-153
The Fate of Anterior Chamber Fluorescein in the Monkey Eye 1. The Anterior Chamber Outflow Pathways STEVEN
H.
SHERMAX*:
KEITH
GREEN-t
BXD
ALAPI'M.
LATIES"
*Department of 0 p hthalmology, Scheie Eye Institute, University of Pennsylvania Medical School, Phildelphi~, Pa 19104, U.S.A. and tDepartnaents of Ophthalmology and Physiology, Me&ml College of Georgia, Augusta, Ga 30901: U.S.A. (Received 28 Sovernber
1977, Sew
York)
As defined by freeze-dry tissue techniques, there are two distinct routes by which fluorescein leaves the anterior chamber of the living rhesus monkey eye. I. The conventional pathway: Within five minutes of the initiation of fluorescein perfusion through the anterior chamber, the conventional pathway filled. Tracer passed through the trabecular meshwork into Schlemm’s canal, and flowed back within scleral and episcleral venous derivatives to enter large veins in the extraocular muscles. II. The uveo-vortex pathway: Fluorescein rapidly moved into the iris stroma and into the anterior part of the ciliary body. The anterior chamber border of the ciliary body is little more than a sparse mesh from which fluorescein entered directly into the substance of the anterior ciliary muscle. Fluorescein penetrated blood vessels in the iris stroma and in the anterior ciliary body from where it could be traced posteriorly to the equatorial choroid and vortex veins. The specific sequential tissue distribution of fluorescein strongly supports the existence of a uveo-vortex pathway for aqueous outflow. Key words: monkey; anterior chamber; aqueous humor; fluorescein; tracer outflow pathway; Schlemm’s canal; uveo-vortex.
1. Introduction The routes by which aqueous humor leaves the anterior chamber have been the subject of numerous studies. For instance, in 1937 von Seidel introduced a solution of indigo-carmine dye into the anterior chamber of the rabbit and later noted the presenceof the dye in the episcleral veins as well as in the iris and ciliary body (von Seidel, 1937). As a result of this and subsequent studies, a conventional outflow pathway through the trabecular meshwork, Schlemm’scanal, intrascleral and episcleral veins to the orbital venous system has been defined (Fine, 1964; Bill, 1965a; Jocson and Grant, 1965; Jocson and Sears, 1969). In addition, just as in Seidel’s study, there have been repeated suggestionsover the years for an alternative outflow pathway. For instance, based on the observation that ferritin injected into the anterior chamber enters not only the conventional pathway but also the ciliary body and iris, Fine suggestedthe existence of a second outflow pathway. Such work was greatly extended and refined by Bill who indicated the existence of a two-part, alternative pathway: one that had as its first part the movement of anterior chamber fluids into the uvea and as its second part their subsequent passageout of the eye through the sclera. He termed this the uveoscleral pathway (Bill, 1965a, b). Reprint U.S.A.
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S. H. SHERMAN,
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Despite their number, all previous histological studies have been open to question. In the main this has been due to the possibility of post mortem tracer diffusion and translocation. Since many water soluble compounds and many particles arc mobile during normal histological fixation and preparation, poet mortem artifact is an important consideration in all such studies. In the present report an attempt is made to improve upon prior studies by using quick-freezing and freeze-drying of whole eyes to hinder post mortem movement of a water soluble molecule. Fluorescein was the marker chosen due to its inherent fluorescent character and the sensitivity with which it can be detected.
2. Materials and Methods Six adult rhesus monkeys (M. mula~cc) were anesthetized with intramuscular phen cyclidine (1.25 mg/kg) and intraperitoneal urethane (1 g/kg) and were seated upright in a restraining device. Supplemental urethane was given, when required, to maintain a. suitable depth of anesthesia. Following exposure of the temporal and frontal aspect,s of the eye by removal of bone, two drops of tetracaine were applied to each eye. Each anterior chamber was eannulated with two 22 gauge needles inserted obliquely through the cornea. The needles were connected to matched infusion and withdrawal syringes of a Harvard infusion pump. By this means, the rate of inflow of tracer solution was precisely matched to the rate of aqueous humor withdrawal. The stable, recorded intraocular pressure was 17.4kO.8 mmHg (S.E.M.) for all 12 eyes with the eye connected only to the transducer. The intraocular pressure which was recorded at the end of a 10 min stabilization period was maintained throughout the experiment by means of a constant pressure reservoir connected into the inflow side of the system, with continuous measurement using a Sanborn transducer and a recorder. Normal saline solution was dripped onto the surface of the cornea to prevent drying. The eyes were used sequentially. Perfusion was begun 10 min after cannulation at which time 0.1% sodium fluorescein in normal saline was exchanged for aqueous humor at a rate of 135 pl/min for the first min and at a rate of 34 &min for the duration of the experiment. The initial, high rate of exchange replaced a large proportion of aqueous humor so that sufficient fluorescein was present in the eye to readily identify exit pathways filling early during perfusion. Blue light and 4x loupes were used to search for tracer leakage around the needles during perfusion; none was found. The perfusion lasted 5, 10, 15, 18, 30 or 60 min in different experiments. At the conclusion of each experiment, the inflow and outflow connecting tubes were clamped close to the needles and eyes were rapidly enucleated. They were then immediately frozen in isopentane cooled to -110°C in a bath of liquid nitrogen. No more than 1 min elapsed from the beginning of enucleation to the immersion in isopentane. The eyes were stored over dry ice and then in liquid nitrogen. Thereafter, each eye was freeze-dried at -35°C for 5 weeks. Once dry, the eye was opened and examined grossly. When this was complete, tissue wedges were embedded in paraffin, sectioned (12 pm), and examined with a Zeiss fluorescence microscope with a Zeiss #47 yellow filter. Two of the 12 eyes were lost to the study because of technical accidents during processing.
3. Results Gross exumirution In non-perfused, freeze-dried eyes the aqueous humor remnant is a fine white powder, whereas in experimental eyes the powder varied in color from a pale yellow to a deep orange-red (low to high fluorescein concentration, respectively) depending
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upon its location when examined under daylight. Every experimental eye had a high concentration of fluorescein (orange powder) in the anterior chamber. Two of 12 eyes (one 10 min and one 15 min experiment) had no visible fluorescein in the posterior chamber (but had white powder); several others contained yellow powder which diminished in color intensity outwards and post,eriorly from the pupillary border, providing clear evidence that fluorescein had diffused into the posterior chamber through the pupil. The 60 min perfusion specimen revealed a posterior chamber fluorescein color equal to that of the anterior chamber. To ensure that the reported anterior chamber outflow pathways accurately reflect movement of tracer exclusively from the anterior chamber, rather than a combination of movements originating from both chambers (since the ciliary epithelium may itself remove fluorescein from the posterior chamber), the major pathway descriptions are based solely on specimens in which it was evident that fluorescein had not reached the surface of the ciliary body by access through the posterior chamber.
FIG. 1. Limbus. The trabecular meshwork (dark arrow), Schlemrn’s canal, peritrabecular limbus and scleral spur all contain fluorescein. The episcleral limbal vessels (white arrow) also contain I fluores’ cein. stroma. The scleral zone between peritrabecular Some t xacer has leaked into the adjacent limbus and episolel ral limbus is not stained. SS, scleral spur. C, cornea. (15 min perfusion, x 40).
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Microscopic
8. H. SHERMAN,
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A. M. LATIES
examination
All localizations were performed using the fluorescence microscope. To ascertain sensitivity, serial dilutions of fluorescein were incubated with fresh rabbit choroitl for 20 min. After subsequent processing, it was found that concentrations of 3 x 10 m5>i were still visible. In the present study fluorescein was easily distinguished as it fluoresces in the yellow while the native autofluorescence of ocular tissues. when viewed through a Zeiss 47 barrier filter, is generally blue. Comea. The cornea1en.dothelium, though difficult to seebecauseof its fine structure, did, in several instances, clearly contain fluorescein. After 5 min of anterior-chamber perfusion, a brilliant yellow band had moved forward acrossthe posterior two-thirds of the cornea towards the epithelium. In 10 min, the entire cornea1 thickness was stained with the exception of the epithelinm. which. in most cases,remained fluorescein free.
F IC. 2. (Jiliary body and sclera just behind limbus. The ciliary muscle is diffusely stained by fluo rescein; the sclera (8) is not. Fluorescein-containing interbundle spaces carry it beyond the diffusely stained port ;ion of ciliary body. An arrow points to one of these spsces. (15 min perfusion, x 63).
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IAmbus. In all experiments, the entire trabecular meshwork, the wall of Schlemm’s canal and the immediately adjacent cornea1 stroma and sclera were diffusely stained by fluorescein. When eyes were perfused for less than 60 min, the deep limbus was separated from the episcleral limbus by a mid-zone of unstained sclera. Intrascleral vessels traversing this unstained zone were of three types: (a) blood-free and containing only fluorescein; (b) lacking fluorescein and containing blood only (usually small arterioles) ; and (c) containing a mixture of fluorescein and blood. In the episcleral limbus, all three blood vessel types were again recognizable. Often dye-containing vessels were surrounded by a yellow stain indicating outward leakage of fluorescein (Fig. 1). In the eye perfused for 60 min, the entire limbal thickness was stained yellow by fluorescein.
FJG. 3. Sclera over pars plana. The anterior ciliary blood vessels lie b&Keen the sciera and episclera. The walls of the veins are stained by fluorescein. A small amount of tracer is visualized in the adjacent tissue. A, anterior ciliary artery: E. episclera: S, sclcra; V, anterior ciliary rein (15 min perfusion, z 63).
X&m rend episclera. Diffuse fluorescein-staining of the sclera ended at the scleral spur; it did not extend posteriorly as far as fluorescein within the ciliary body or choroid. Since the two were not coextensive, a sharp border between the yellow (fluoreecein) ciliary muscle and the blue (autofluorescence) sclera was often seen (Fig. 2). Th e supraciliary spaceand the adjacent eiliary body could not; he differentiated in our preparations on the basis of fluorescein intensity. 9 moderate number of fluorescein-filled blood vesselswere found in the episcleral connective tissue. Occasionally, tracer leaked into the adjacent sclera,causing a yellow halo on the blood vessel (Fig. 3). Further posteriorly, large fluorescein-tiled veins could, on occasion, be directly traced into extraocu.lar muscles. Cross sections of extraocular musclesfrom one 30 min eye clearly revealed fluorescein within anterior ciliary veins with sometracer diffusing out of the vein to stain t,he surrounding muscle:
PIG. 4. Cross section of an c~st,roocular muscle near its insertiol1. l‘he antrrior prescein while the anterior ciliary altery does not (3~ nlitl l)ct,fusion, :< 63).
FIG. 5. Iris. Fluorescein permeates the iris stroma fluorescein (arrows). AC, anterior chamber (15 min
and stains the blood perfusion, x 63).
vessel walls.
ciliary
win
rontai
Note intravascular
.ns
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FIG. 6. Ciliary and erythrocytes. .< 10).
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body. The anterior ciliary muscle is diffusely yellow. A basal rein is filled with fluorescein Arrow points to iris root. PC, posterior chamber; CNI, ciliary muscle (15 min perfusion,
FIG. 7. Fluorescein-filled ciliary muscle vein (V). immersionl.
interbundle spaces extend throughout Irregular, dark objects are melanocytes
the ciliary muscle and surround a (51). (30 min perfusion, >; 160, oil
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(Fig. 4). Fluorescein was rarely found in episcleral veins behind the point at which the anterior ciliary vein3 entered the extraocular muscles, implying that most of the episcleral vein drainage occurred through the ant’erior ciliary veins to t.hct extfraoculal muscles. No tracer-filled veins were seen passing het\yeen the ciliary muscle and sclera. despite the fact that we specifically tried to find such connections. Iris. The iris stroma was completely infiltrated by fluorescein, from the anterio border layer to the pigment epithelium, in all specimens. This was irrespective of the duration of the perfusion. Although fluorescein was never recognized within the make tracer pigmented epithelium, the dense black nature of tlhis structure woultl detection difficult. Not only were the iris blootl-vessel walls stained with fluorescein but it was seen to be mixed with blood cells within t,hem (Fig. 5). clearly indicating penetration. In seveml t,issue sections, a large vein containing fluorescein could l)e observed at the iris root adjacent to the base of the ciliary body (Fig. 6).
Fm. 8. Ciliary muscle. A large vein contains fluorescein nithin its lumen. ‘l’he vein is stained yell~nv and it is surrounded by interbnntife spaces which also contain fluorescein. Fluo~esccin-colltail,ing interbundle spaces not immediately associat,ed with veins BPC also present. Thr white arrow points to one of these. (60 min perfusion, :C 63).
C’iZkw~ bodg. The face of the cilinry body (next to the root of the iris) and the adjacent longitudinal fibers of the ciliary muscle lvere yellow in all but one of the experimental eyes; these structures were blue in the controls. In general, the longer the perfusion lasted, the further fluorescein wa,s seen to penetrate posteriorly into the ciliary body. Close examination of the ciliary body revealed three distinctive and successive patterns of fluorescein distribution; diffuse, focal and intravawular.
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FLUORESCEIN
The anterior one-third of the ciliary body adjacent to the iris was diffusely stained (Fig 6) ; in a Sex specimens the stain extended as far posteriorly as the pars plana. The diffuse stain was limited to the outer (or more peripheral) half of the
Fro. ,., 63).
9. A basal win
at the OI‘B serrate
FIG. 10. Fluorescein
(OS)
within
conkins
vortex
fluorwcrin.
rein
located
VIT,
inside
vitrrous.
the sclers.
(15 min
perfusion
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ciliary body. The inner half of the ciliary body, that lying closer to the ciliary epithelium, was free of tracer (except that present in blood vessels). Just beyond the diffusely yellow area. focal stain was found in connective tistiue spaces between ciliary muscle fiber l)undles (see Fig. 7), extending posteriorly for a variable distance. Occasionally, there existed a close relationship between ciliary muscle blood vessels and these intermuscle bundle channels wherein numerous yellow-stained channels surround a blood vessel wall. Lastly, fluorescein was frequently observed within blood vessels, mainly in veins (Figs 6 and 8). Suc h veins were most commonly seen in the body of the ciliary muscle or at its base, often being observed as far posterior as the pars plana. In three eyes the dye was seen in veins in the anterior choroid (Pig. 9), in two of these it was followed into the ampulla of a vortex vein (Pig. 10). It, was also sern on occasion in vortex veins outside the scleral wall. No fluorescein was detected in the choroid posterior to vortex veins. In fact, serial sections of the ampullae of the vortex veins of one specimen revealed a remarkable segregation : fluorescein was rea,dilv visible in several anterior radicals and it was not visible in the posterior radicals which drain the back of the eye No fluorescein was visualized anywhere in or on the retina or in or about the optic nerve. Cilicwy processes. When fluorescein within the posterior chamber came within reach, the ciliary processes picked it up. sot only could a yellow cpithelial stain then be seen, but in the 60 min perfusion fluorescein had moved inward from the e-t;ithelium to the ciliary process stroma. 4. Discussion The conventional outflow pathway beginning at the trabecular meshwork and Schlemm’s canal does not account for all aqueous drainage; several authors have shown that substances injected into the anterior chamber penetrate into iris and ciliary body (Fine, 1964; Bill. 1965a, 1967a; Jocson and Grant, 1965; Jocson and Sears, 1969; Ishikawa, 1962). The present study was made to clarify the relationship of the anterior chamber to the uvea. Because of its physical and chemical characteristics (Maurice, 1967) fluorescein is superior to many tracers used in prior studies of aqueous outflow pathways. Its major limitation, however, is that as an organic acid, it can cross many cells by carrier mediat.ed transport. The technique we used (Gersh, 1937; Grayson, Tsukahara and Laties, 1974) limits postmortem diffusion and translocation and can be combined with fluorescence microscopy to enhance sensitivity. Since most ocular tissues fluoresce in the blue under the conditions of our experiment, accurate fluorescein identification is assured. The presence of crisply-margined tracer borders at bloodocular barrier interfaces is perhaps the best assurance that free diffusion is restricted (see Fig. 8). When through ex_ p erimental error such as delayed freezing, inadvertent warming of frozen tissue, exposure of dried tissue to air-an opportunity is afforded for diffusion, blurred borders inevitably are seen. Care was exercised to ensure that the normal ocular physiology was disturbed to the least extent possible in order that the observed pathways would not be a product of experimental manipulation. The macaque monkey was chosen because of the similarity of its eye to that of the human. Anterior segment structure among mammals varies among different species to an extreme clegree (Tripathi, 1974), and, if comparability to the human eye is a consideration, then only species with a closed ciliary cleft and with massive ciliary muscle development yield a valid comparison.
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The major results of our experiments can be summarized as follows: Fluorescein introduced into the anterior chamber of the macaque monkey enters the systemic circulation, not only by passing through Schlemm’s canal, but also by gaining access to uveal blood vessels. By using perfusion times of varying duration and by studying the entire globe, fluorescein could be traced through the conventional outflow pathway and through an unconventional pathway. The latter consisted of a venous drainage network ending in the vortex veins. The borders of the anterior chamber The nature of the iris anterior border layer has been open to question due to its peculiar structure. Based on the present experiments, and the observations by Vrabec (1952) and by Magari (1957) the physiological importance of the front surface of the iris as a barrier is questioned. Once present in the anterior chamber, fluorescein enters the interstices of the iris stroma, the anterior ciliary body and the trabecular meshwork so rapidly that, as measured by our methods, these anatomical entities form, for practical purposes, a single coextensive fluid compartment. Fluorescein, however, is rigorously excluded from the posterior chamber by the pigmented epithelial barrier at the posterior face of the iris. Fluorescein enters the posterior chamber only via the pupil; actually through the small gap between the iris rim and the front surface of the lens. The conventional ou@ow pathway Since fluorescein was visible in the conventional pathway and absent in the ciliary body in two of the three 5 min experiments, it seems reasonable to suggest that the conventional system f?.lls more rapidly than does the uveal system; this observation has been previously made (Bill, 1967a). A schematic diagram of the conventional pathway for fluorescein is shown in Fig. 11: tracer passes sequentially through the
FIG.
11. Schematic
presentation
of the conventional
outflow
path\%--a!: for anterior
chamber
fluorescein.
trabecular meshwork, Schlemm’s canal, intrascleral channels and thence into episcleral veins. From this point fluorescein passes into larger veins in the extraocular muscles to join the orbital circulation. The leakiness of the veins outside the eye leads to a modest diffusion of tracer into surrounding tissues.
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The uveo-vortex outjiou’ ynthwq
The iris stroma in monkeys has been found freely permeable to tracers placed within the anterior chamber (McMaster and Macri, 1968; Saari, 1974; Smelser and Ishikawa, 1962). Jocson and Grant (1965) and Jocson and Sears (1969) have also shown (both in human and monkey eyes) that even a high viscosity silicon polymer injected into the anterior chamber can infiltrate the anterior aspect of the ciliary nrusclc. Further, the penetration of a variety of tracers (indigocarmine (lye, ferritin. thorotrast and inulin) into blood vesselsof the iris and ciliary body has been demonstrated (von Seidel, 1937; Fine, 1964; Ishikawa, 1962; Saari, 1974; Inomata, Bill and Xmelser, 1972; Fowlks and Havener, 1964; Holmberg, 1959; Taniguchi, 1962). while studies using inulin have shown that it passesposteriorly into the suprachoroitl (l&Master and Macri: 1968). Since the opposite is not true, that is. the sameor similar tracers do not leave the blood vesselsof the iris (Von Sallmann and Dillon, 1967; Shakib and Cunha-Vaz, 1966); or of the ciliary muscle (Grayson and Laties, 1971) after intravascular administration. these anterior segment blood vesselsare directIS comparable to retinal blood vessels(C’unha-Vaz and Maurice; 1967). In essenceour report confirms selectedparts of several previous studies. Given the advantage of viewing fluorescein in eyes quick-frozen and then freeze-dried, it is possibleto re-order the aforementioned observations to identify the defining characteristics of a uveo-vortex outflow pathway. The pathwav extends all the way from iris to choroid. Its particulars are as follows : Fluorescein-penetrates blood vesselsin the iris; simultaneously, fluorescein diffusing freely in the interstices of the anterior part of the ciliary muscleenters local blood vessels(Figs 2. 5. i). Thereafter, asthe venous blood flows normally towards the choroicl to leave the eye via vortex veins. tracer from both sources. from iris and from anterior ciliary muscle, mingles together. To the extent that any part of this venous flow leaves the eye through the anterior ciliary complex (for details seeJocson and Grant, 1965 xntl Jocson and Sears. 1969), an intravascular uveo-scleral outflow also ta,kes place. Further, the filling of blood vessels in the ciliary muscle appears to follow a squential pattern: initial general diffusion of the tracer within ciliary muscle tissue is folio\\-ed by k%aliZatioli in connective ti%uC s])acW. and filially, p’I?k’&ti(Jll illtO 1,lood vnW?ls.
In 1964 Fine clearly demonstrated the passageof large moleculesfrom the anterior chamber into the ciliary body. Bill and his collaborators (1965a; 1966; 196ia,b; Inomata. Bill and Smelser (1972)) further demonstrated the passageof large molecules from the anterior chamber into the ciliary body and then into the supraciliary space. From there, in their experiments, the tracers apparently passed out directly through the sclera (Bill, 1965b) and accumulated in the episcleral tissueswhich are drained by conjunctival lymph vessels. The fact that tracers of differing diffusion coefficients movecl along this route at roughly the samerate argued that their passage was due to a bulk fluid flow of acpieoushumor. This route was called the uveo-sclcral pathway, since it invoked both uveal and trans-scleral pathways. Physiological experiments in monkeys showed that this route accounted for about 20% of total outflow at normal intraocular pressure. Flow through the uveo-scleral pathway was found to change little with changesin intraocular pressure,but to be nearly abolished by pilocarpine (Bill, 1967a).
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FLUORESCEIN
In our study fluorescein was observed to leave the anterior chamber primarily by the conventional and uveo-vortex pathways. In all eyes, staining of the sclera mainly ~~~~rred in the immediate vicinity of small, trans-scleral vessels which leaked dye near the limbus. The uveo-scleral pathway and the uveo-vortex pathway, as proposed, are essentially the same within the uvea since, in each, tracer moves from the anterior chamber into the ciliarg body. However, the tracer following the uveo-scleral route is then thought to pass out of the eye through the sclera: whereas that following the uvoovortex route leaves the eye by the vortex veins. Rntmme
of jhorescein
into the weal
venous systenl
The techniques employed do not permit a critical evaluation among competing mechanisms through which fluorescein could enter the uveal blood vessels. Thus without a quantitative measure or the use of inhibitor (Maurice, 19671,our results do not discriminate between the two chief means by which fluorescein could enter the uvoal circulation-active transport or passive ultrafiltration. Fortunately, this pro1A.ln has recently been addressed by Pederson, Gaasterland and XacLellan (1%) who: in comparing the passageof fluorescein to t’hat of lz51-labeledalbumin from t’he anterior chamber into the uveal circulation and vortex veins, found the t1v-o diffcro(l in such a manner to indicate a major role for passivereabsorption of materials. depending on their size. Tracer concentration \~,s measured in a cannulated vortex vein and compared with concentrations in the svst,emic bloodstream. The same
PI,:.
13. Schematic
presentation
of the uveo-vortex
outflow
pathway
for anterior
chamber
fluoreswin.
authors by showing a pressure dependent increase in fluorescein and albumin concentrations in the uveal circulation have furnished a second strand of evidence in favor of a passive ultrafiltration as a mechanism for fluid reabsorption. In these experiments it was found that the contribution of uveo-vortex outflow to total outflow was about lo?;, a value lower than that indicated from previous studies. The anxtSornicalstudies reported here have, therefore, been substantiated by physiological evidence (Pederson et al., 1977). Since as already noted there are strong parallels beta-een the blood vesselsof the retina and iris, the active transport of fluorescein from anterior segment structures is not entirely ruled out (Maurice, 1967). In fact, the participation of sucha mechanism to explain some part of the movement of fluorescein into iris blood vesselsremains an open possibility.
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A. 11. LATIES
In conclusion, we believe there are two separate pathways by which fluorescein leaves the anterior chamber. Iu the conventional pathway (Fig. 11) fluorescein l)as!;es sequentially through the tral)eculaa nleshwork. Schlemm’s ca,nal. t’be aqrueons ant1 cpiscleral veins, the anterior ciliarg veins ant1 hence into the systemic circulation. These vessels often are permeable to t#racer, leading t’o its diffusion into extravascrilar tissues. In the uveo-vortex pathway fluorescein enters the iris and the ciliary muscle directly from the anterior chamber, In the iris, tracer enters blood vessels (Fig. 12). Simultaneously, tracer diffusing posteriorly (Fig. 12) within the ciliary muscle also penetrates blood vessels. Thereafter. a significant amount of fluorescein passes out of the eye via normal nveal drainage through the vortex veins. No general trans.scleral passage of fluoresoein has been found and thus we cannot confirm the existence of a separate extravascular uveo-sclercrl pathway. Since further experiments using fluorescein-labeletl dextran have indicated generally similar patterns of distribution to those found with fluorescein alone, we suggest that even large molecular weight (80 000 daltons) molecules can enter the blood vessels of the iris and ciliary body. Some degree of active transport of free fluorescein into the uveo-vortex pathway camrot be excluded by the present experiments. ACKNOWLEDGMENTS
Supported in part by U.S. Public Health Service Research Grants EY 01413, EY 01329 and EY 01194 from the National Eye Institute. We thank Mm Karen Bowman and Miss Alice McGlinn for their valuable technical assistance, MIX I. Prior for secretarial assistance and Drs J. M. Kling and R. G. Shimp and Mr R. Morrison for their help with animal preparation and anesthesia. We also thank Dr Anders Bill for valuable criticism and for advice on the experiments. REFERENCES 9. (1965a). The protein exchange in the eye with aspects on conventional and uveo-scleral bulk drainage of aqueous humor in primates. In Symposium on Eye Structure, II. (Ed. Rohen, J. W.). Pp. 313-20. Schattauer-Verlag, Stuttgart. Bill, A. (1965b). The aqueous humor drainage mechanism in the cynomolgus monkey (fllacacn irus) with evidence for unconventional routes. Invest. Ophthalmol. 4, 911-9. Bill, A. (1966). Formation and drainage of aqueous humor in cats. Exp. Eye Res. 5, 185-90. Bill, 8. (1967a). Effects of atropine and pilocarpine on aqueous humor dynamics in cynomolgus monkeys. Exp. Eye Res. 6, 120-5. Bill, A. (19’76b). Further st’udies on the influence of the intraocular pressure on aqueous humor dynamics in the cynomolgus monkey. Invest. Ophthalmol. 6, 364-72. Cunha-Vaz, J. G. and Maurice, D. M. (1967). The active transport of fluorescein by the rctinnl vessels and the retina. J. Physiol. (Lord) 191, 467-86. Fine, B. S. (1964). Observations on the drainage angle in man and rhesus monkey: a concept of the pathogen&s of chronic simple glaucoma. Inwest. Ophthnlmol. 3, 609-46. Fowlks, W. L. and Havener, V. R. (1964). Aqueous flow into the perivascular space of the rabbit, ciliary body. Invest Ophthalmol. 3, 374-83. Gersh, I. (1937). Improved histochemical methods for chloride, phosphate, carbonate and potassium applied to skeletal muscles. Anat. Rec. 70, 311-29. Grayson, M. C. and Laties, A. M. (1971). Ocular localization of sodium fluorescein. Arch. Ophthdmol. 85,600-9. Grayson, M., Tsukahara, S. and Laties. A. (1974). Tissue localization in the rabbit and monkey eye of intravenously administered fluorescein. In Fluoresce& Angiography (Ed. Shmizu, K.). Pp. 23546. Igaku Shoin Ltd., Tokyo. Holmberg, A. (1959). The ultrastructure of the capillaries in the ciliary body. Arch. Ophthalmol 62, 94951. Bill,
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Inomata, H., Bill, A. and Smelser, G. K. (1972). Unconventional routes of aqueous humor outflow in cynomolgus monkey (.Macacu irus) Am. J. Ophthalmol. 73, 893-907. Ishikawa, T. (1962). Fine structure of the human ciliary muscle. Invest. Ophthalmol. 1, 587-608. Jocson, V. L. and Grant, W. M. (1965). Interconnections of blood vessels in human eyes. Arch. Ophthalm,ol. 73,707-20. Jocson, V. L. and Sears, M. L. (1969). Ch annels of aqueous outflow and related blood vessels. II. Cercapithecus etbiops, Ethiopian green or green vervet. Arch. Ophthulmd. 81, 24443. Magari, S. (1957). Zur Struktur der Stromas der Iris and zur Frage einea Endothels an Ihrer Vorderflache, Albrecht von Graefes Arch. Ophthalmol. 159, 257-76. Maurice, D. M. (1967). The use of fluorescein in ophthalmological research. Invest. Ophthalmol. 6, 464-77. McMaster, P. R. B. and Macri, F. J. (1968). Secondary aqueous humor outflow pathways in the rabbit, cat and monkey. Arch. Ophthalmol. 79, 297-303. Pederson, J. E., Gaasterland, D. E. and MacLellan, H. M. (1977). Uveo-scleral aqueous outflow in the rhesus monkey: importance of uveal absorption. Ivwest. Ophthalmol. 16, 1008-17. Saari, M. (1974). Morphological basis of diffusion through t,he iris vessels in the dynamics of aqueous humor. Acta. Ophthalm.ol. Suppl. 123,26-M. Shakib, M. and Cunha-Vaz, J. G. (1966). Studies on the permeability of t.he blood-retinal barrier. 1V. Junctional complexes of the retinal vessels and their role in permeability of t#he bloodretinal barrier. Exp. Eye Res. 5,229. Smelser, G. K. and Ishikawa, T. (1962). Investigation on the porosity of the iris. 19th Congress of Ophthalmol. 1. 612-23. Taniguchi, Y. (1962). Fine structure of the blood vessels in the ciliary body. Jap. J. Ophthrtlmol. 6,93-103. Tripathi, R. (‘. (1974). Comparative physiology and anatomy of the outflow pathway. The Eye. Vol. V, (Eds Davson, H. and Graham, L. T., Jr.). Pp. 163-336. Academic Press, N.Y. Von Sallmanrl. L. and Dillon, B. (1967). The effect of diisopropylfluorophosphate on the capillaries of the anterior segment of the eye in rabbits. Am. J. Ophthalm.ol. 30, 1244. von Seidel. E. (1937). Methoden zur untersuchung des intraokularen flussigkeitswechels. In Handbuch der Biologischen Arbeitsmethoden, Abt. V: Methoden zum Studium der Funktionen der einzelnen Organe des tierischen Organismus. P. 1069. Vrabec, F. (19.52). The anterior superficial endothelium of the human iris. Ophfhalmolog%ca. 123, “(l--30.
The Fate of Anterior Chamber Fluorescein in the Monkey Eye 1. The Anterior Chamber Outflow Pathways STEVEN
H.
SHERMAX*:
KEITH
GREEN-t
BXD
ALAPI'M.
LATIES"
*Department of 0 p hthalmology, Scheie Eye Institute, University of Pennsylvania Medical School, Phildelphi~, Pa 19104, U.S.A. and tDepartnaents of Ophthalmology and Physiology, Me&ml College of Georgia, Augusta, Ga 30901: U.S.A. (Received 28 Sovernber
1977, Sew
York)
As defined by freeze-dry tissue techniques, there are two distinct routes by which fluorescein leaves the anterior chamber of the living rhesus monkey eye. I. The conventional pathway: Within five minutes of the initiation of fluorescein perfusion through the anterior chamber, the conventional pathway filled. Tracer passed through the trabecular meshwork into Schlemm’s canal, and flowed back within scleral and episcleral venous derivatives to enter large veins in the extraocular muscles. II. The uveo-vortex pathway: Fluorescein rapidly moved into the iris stroma and into the anterior part of the ciliary body. The anterior chamber border of the ciliary body is little more than a sparse mesh from which fluorescein entered directly into the substance of the anterior ciliary muscle. Fluorescein penetrated blood vessels in the iris stroma and in the anterior ciliary body from where it could be traced posteriorly to the equatorial choroid and vortex veins. The specific sequential tissue distribution of fluorescein strongly supports the existence of a uveo-vortex pathway for aqueous outflow. Key words: monkey; anterior chamber; aqueous humor; fluorescein; tracer outflow pathway; Schlemm’s canal; uveo-vortex.
1. Introduction The routes by which aqueous humor leaves the anterior chamber have been the subject of numerous studies. For instance, in 1937 von Seidel introduced a solution of indigo-carmine dye into the anterior chamber of the rabbit and later noted the presenceof the dye in the episcleral veins as well as in the iris and ciliary body (von Seidel, 1937). As a result of this and subsequent studies, a conventional outflow pathway through the trabecular meshwork, Schlemm’scanal, intrascleral and episcleral veins to the orbital venous system has been defined (Fine, 1964; Bill, 1965a; Jocson and Grant, 1965; Jocson and Sears, 1969). In addition, just as in Seidel’s study, there have been repeated suggestionsover the years for an alternative outflow pathway. For instance, based on the observation that ferritin injected into the anterior chamber enters not only the conventional pathway but also the ciliary body and iris, Fine suggestedthe existence of a second outflow pathway. Such work was greatly extended and refined by Bill who indicated the existence of a two-part, alternative pathway: one that had as its first part the movement of anterior chamber fluids into the uvea and as its second part their subsequent passageout of the eye through the sclera. He termed this the uveoscleral pathway (Bill, 1965a, b). Reprint U.S.A.
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to: Dr A. M. Laties,
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$01.00/O
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Despite their number, all previous histological studies have been open to question. In the main this has been due to the possibility of post mortem tracer diffusion and translocation. Since many water soluble compounds and many particles arc mobile during normal histological fixation and preparation, poet mortem artifact is an important consideration in all such studies. In the present report an attempt is made to improve upon prior studies by using quick-freezing and freeze-drying of whole eyes to hinder post mortem movement of a water soluble molecule. Fluorescein was the marker chosen due to its inherent fluorescent character and the sensitivity with which it can be detected.
2. Materials and Methods Six adult rhesus monkeys (M. mula~cc) were anesthetized with intramuscular phen cyclidine (1.25 mg/kg) and intraperitoneal urethane (1 g/kg) and were seated upright in a restraining device. Supplemental urethane was given, when required, to maintain a. suitable depth of anesthesia. Following exposure of the temporal and frontal aspect,s of the eye by removal of bone, two drops of tetracaine were applied to each eye. Each anterior chamber was eannulated with two 22 gauge needles inserted obliquely through the cornea. The needles were connected to matched infusion and withdrawal syringes of a Harvard infusion pump. By this means, the rate of inflow of tracer solution was precisely matched to the rate of aqueous humor withdrawal. The stable, recorded intraocular pressure was 17.4kO.8 mmHg (S.E.M.) for all 12 eyes with the eye connected only to the transducer. The intraocular pressure which was recorded at the end of a 10 min stabilization period was maintained throughout the experiment by means of a constant pressure reservoir connected into the inflow side of the system, with continuous measurement using a Sanborn transducer and a recorder. Normal saline solution was dripped onto the surface of the cornea to prevent drying. The eyes were used sequentially. Perfusion was begun 10 min after cannulation at which time 0.1% sodium fluorescein in normal saline was exchanged for aqueous humor at a rate of 135 pl/min for the first min and at a rate of 34 &min for the duration of the experiment. The initial, high rate of exchange replaced a large proportion of aqueous humor so that sufficient fluorescein was present in the eye to readily identify exit pathways filling early during perfusion. Blue light and 4x loupes were used to search for tracer leakage around the needles during perfusion; none was found. The perfusion lasted 5, 10, 15, 18, 30 or 60 min in different experiments. At the conclusion of each experiment, the inflow and outflow connecting tubes were clamped close to the needles and eyes were rapidly enucleated. They were then immediately frozen in isopentane cooled to -110°C in a bath of liquid nitrogen. No more than 1 min elapsed from the beginning of enucleation to the immersion in isopentane. The eyes were stored over dry ice and then in liquid nitrogen. Thereafter, each eye was freeze-dried at -35°C for 5 weeks. Once dry, the eye was opened and examined grossly. When this was complete, tissue wedges were embedded in paraffin, sectioned (12 pm), and examined with a Zeiss fluorescence microscope with a Zeiss #47 yellow filter. Two of the 12 eyes were lost to the study because of technical accidents during processing.
3. Results Gross exumirution In non-perfused, freeze-dried eyes the aqueous humor remnant is a fine white powder, whereas in experimental eyes the powder varied in color from a pale yellow to a deep orange-red (low to high fluorescein concentration, respectively) depending
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upon its location when examined under daylight. Every experimental eye had a high concentration of fluorescein (orange powder) in the anterior chamber. Two of 12 eyes (one 10 min and one 15 min experiment) had no visible fluorescein in the posterior chamber (but had white powder); several others contained yellow powder which diminished in color intensity outwards and post,eriorly from the pupillary border, providing clear evidence that fluorescein had diffused into the posterior chamber through the pupil. The 60 min perfusion specimen revealed a posterior chamber fluorescein color equal to that of the anterior chamber. To ensure that the reported anterior chamber outflow pathways accurately reflect movement of tracer exclusively from the anterior chamber, rather than a combination of movements originating from both chambers (since the ciliary epithelium may itself remove fluorescein from the posterior chamber), the major pathway descriptions are based solely on specimens in which it was evident that fluorescein had not reached the surface of the ciliary body by access through the posterior chamber.
FIG. 1. Limbus. The trabecular meshwork (dark arrow), Schlemrn’s canal, peritrabecular limbus and scleral spur all contain fluorescein. The episcleral limbal vessels (white arrow) also contain I fluores’ cein. stroma. The scleral zone between peritrabecular Some t xacer has leaked into the adjacent limbus and episolel ral limbus is not stained. SS, scleral spur. C, cornea. (15 min perfusion, x 40).
162
Microscopic
8. H. SHERMAN,
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A. M. LATIES
examination
All localizations were performed using the fluorescence microscope. To ascertain sensitivity, serial dilutions of fluorescein were incubated with fresh rabbit choroitl for 20 min. After subsequent processing, it was found that concentrations of 3 x 10 m5>i were still visible. In the present study fluorescein was easily distinguished as it fluoresces in the yellow while the native autofluorescence of ocular tissues. when viewed through a Zeiss 47 barrier filter, is generally blue. Comea. The cornea1en.dothelium, though difficult to seebecauseof its fine structure, did, in several instances, clearly contain fluorescein. After 5 min of anterior-chamber perfusion, a brilliant yellow band had moved forward acrossthe posterior two-thirds of the cornea towards the epithelium. In 10 min, the entire cornea1 thickness was stained with the exception of the epithelinm. which. in most cases,remained fluorescein free.
F IC. 2. (Jiliary body and sclera just behind limbus. The ciliary muscle is diffusely stained by fluo rescein; the sclera (8) is not. Fluorescein-containing interbundle spaces carry it beyond the diffusely stained port ;ion of ciliary body. An arrow points to one of these spsces. (15 min perfusion, x 63).
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IAmbus. In all experiments, the entire trabecular meshwork, the wall of Schlemm’s canal and the immediately adjacent cornea1 stroma and sclera were diffusely stained by fluorescein. When eyes were perfused for less than 60 min, the deep limbus was separated from the episcleral limbus by a mid-zone of unstained sclera. Intrascleral vessels traversing this unstained zone were of three types: (a) blood-free and containing only fluorescein; (b) lacking fluorescein and containing blood only (usually small arterioles) ; and (c) containing a mixture of fluorescein and blood. In the episcleral limbus, all three blood vessel types were again recognizable. Often dye-containing vessels were surrounded by a yellow stain indicating outward leakage of fluorescein (Fig. 1). In the eye perfused for 60 min, the entire limbal thickness was stained yellow by fluorescein.
FJG. 3. Sclera over pars plana. The anterior ciliary blood vessels lie b&Keen the sciera and episclera. The walls of the veins are stained by fluorescein. A small amount of tracer is visualized in the adjacent tissue. A, anterior ciliary artery: E. episclera: S, sclcra; V, anterior ciliary rein (15 min perfusion, z 63).
X&m rend episclera. Diffuse fluorescein-staining of the sclera ended at the scleral spur; it did not extend posteriorly as far as fluorescein within the ciliary body or choroid. Since the two were not coextensive, a sharp border between the yellow (fluoreecein) ciliary muscle and the blue (autofluorescence) sclera was often seen (Fig. 2). Th e supraciliary spaceand the adjacent eiliary body could not; he differentiated in our preparations on the basis of fluorescein intensity. 9 moderate number of fluorescein-filled blood vesselswere found in the episcleral connective tissue. Occasionally, tracer leaked into the adjacent sclera,causing a yellow halo on the blood vessel (Fig. 3). Further posteriorly, large fluorescein-tiled veins could, on occasion, be directly traced into extraocu.lar muscles. Cross sections of extraocular musclesfrom one 30 min eye clearly revealed fluorescein within anterior ciliary veins with sometracer diffusing out of the vein to stain t,he surrounding muscle:
PIG. 4. Cross section of an c~st,roocular muscle near its insertiol1. l‘he antrrior prescein while the anterior ciliary altery does not (3~ nlitl l)ct,fusion, :< 63).
FIG. 5. Iris. Fluorescein permeates the iris stroma fluorescein (arrows). AC, anterior chamber (15 min
and stains the blood perfusion, x 63).
vessel walls.
ciliary
win
rontai
Note intravascular
.ns
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FIG. 6. Ciliary and erythrocytes. .< 10).
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body. The anterior ciliary muscle is diffusely yellow. A basal rein is filled with fluorescein Arrow points to iris root. PC, posterior chamber; CNI, ciliary muscle (15 min perfusion,
FIG. 7. Fluorescein-filled ciliary muscle vein (V). immersionl.
interbundle spaces extend throughout Irregular, dark objects are melanocytes
the ciliary muscle and surround a (51). (30 min perfusion, >; 160, oil
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8. H. SHERMAK.
K. GKEES
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A. M. LATIES
(Fig. 4). Fluorescein was rarely found in episcleral veins behind the point at which the anterior ciliary vein3 entered the extraocular muscles, implying that most of the episcleral vein drainage occurred through the ant’erior ciliary veins to t.hct extfraoculal muscles. No tracer-filled veins were seen passing het\yeen the ciliary muscle and sclera. despite the fact that we specifically tried to find such connections. Iris. The iris stroma was completely infiltrated by fluorescein, from the anterio border layer to the pigment epithelium, in all specimens. This was irrespective of the duration of the perfusion. Although fluorescein was never recognized within the make tracer pigmented epithelium, the dense black nature of tlhis structure woultl detection difficult. Not only were the iris blootl-vessel walls stained with fluorescein but it was seen to be mixed with blood cells within t,hem (Fig. 5). clearly indicating penetration. In seveml t,issue sections, a large vein containing fluorescein could l)e observed at the iris root adjacent to the base of the ciliary body (Fig. 6).
Fm. 8. Ciliary muscle. A large vein contains fluorescein nithin its lumen. ‘l’he vein is stained yell~nv and it is surrounded by interbnntife spaces which also contain fluorescein. Fluo~esccin-colltail,ing interbundle spaces not immediately associat,ed with veins BPC also present. Thr white arrow points to one of these. (60 min perfusion, :C 63).
C’iZkw~ bodg. The face of the cilinry body (next to the root of the iris) and the adjacent longitudinal fibers of the ciliary muscle lvere yellow in all but one of the experimental eyes; these structures were blue in the controls. In general, the longer the perfusion lasted, the further fluorescein wa,s seen to penetrate posteriorly into the ciliary body. Close examination of the ciliary body revealed three distinctive and successive patterns of fluorescein distribution; diffuse, focal and intravawular.
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FLUORESCEIN
The anterior one-third of the ciliary body adjacent to the iris was diffusely stained (Fig 6) ; in a Sex specimens the stain extended as far posteriorly as the pars plana. The diffuse stain was limited to the outer (or more peripheral) half of the
Fro. ,., 63).
9. A basal win
at the OI‘B serrate
FIG. 10. Fluorescein
(OS)
within
conkins
vortex
fluorwcrin.
rein
located
VIT,
inside
vitrrous.
the sclers.
(15 min
perfusion
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8. H. SHEIWAS.
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AND
A. M. LATIES
ciliary body. The inner half of the ciliary body, that lying closer to the ciliary epithelium, was free of tracer (except that present in blood vessels). Just beyond the diffusely yellow area. focal stain was found in connective tistiue spaces between ciliary muscle fiber l)undles (see Fig. 7), extending posteriorly for a variable distance. Occasionally, there existed a close relationship between ciliary muscle blood vessels and these intermuscle bundle channels wherein numerous yellow-stained channels surround a blood vessel wall. Lastly, fluorescein was frequently observed within blood vessels, mainly in veins (Figs 6 and 8). Suc h veins were most commonly seen in the body of the ciliary muscle or at its base, often being observed as far posterior as the pars plana. In three eyes the dye was seen in veins in the anterior choroid (Pig. 9), in two of these it was followed into the ampulla of a vortex vein (Pig. 10). It, was also sern on occasion in vortex veins outside the scleral wall. No fluorescein was detected in the choroid posterior to vortex veins. In fact, serial sections of the ampullae of the vortex veins of one specimen revealed a remarkable segregation : fluorescein was rea,dilv visible in several anterior radicals and it was not visible in the posterior radicals which drain the back of the eye No fluorescein was visualized anywhere in or on the retina or in or about the optic nerve. Cilicwy processes. When fluorescein within the posterior chamber came within reach, the ciliary processes picked it up. sot only could a yellow cpithelial stain then be seen, but in the 60 min perfusion fluorescein had moved inward from the e-t;ithelium to the ciliary process stroma. 4. Discussion The conventional outflow pathway beginning at the trabecular meshwork and Schlemm’s canal does not account for all aqueous drainage; several authors have shown that substances injected into the anterior chamber penetrate into iris and ciliary body (Fine, 1964; Bill. 1965a, 1967a; Jocson and Grant, 1965; Jocson and Sears, 1969; Ishikawa, 1962). The present study was made to clarify the relationship of the anterior chamber to the uvea. Because of its physical and chemical characteristics (Maurice, 1967) fluorescein is superior to many tracers used in prior studies of aqueous outflow pathways. Its major limitation, however, is that as an organic acid, it can cross many cells by carrier mediat.ed transport. The technique we used (Gersh, 1937; Grayson, Tsukahara and Laties, 1974) limits postmortem diffusion and translocation and can be combined with fluorescence microscopy to enhance sensitivity. Since most ocular tissues fluoresce in the blue under the conditions of our experiment, accurate fluorescein identification is assured. The presence of crisply-margined tracer borders at bloodocular barrier interfaces is perhaps the best assurance that free diffusion is restricted (see Fig. 8). When through ex_ p erimental error such as delayed freezing, inadvertent warming of frozen tissue, exposure of dried tissue to air-an opportunity is afforded for diffusion, blurred borders inevitably are seen. Care was exercised to ensure that the normal ocular physiology was disturbed to the least extent possible in order that the observed pathways would not be a product of experimental manipulation. The macaque monkey was chosen because of the similarity of its eye to that of the human. Anterior segment structure among mammals varies among different species to an extreme clegree (Tripathi, 1974), and, if comparability to the human eye is a consideration, then only species with a closed ciliary cleft and with massive ciliary muscle development yield a valid comparison.
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The major results of our experiments can be summarized as follows: Fluorescein introduced into the anterior chamber of the macaque monkey enters the systemic circulation, not only by passing through Schlemm’s canal, but also by gaining access to uveal blood vessels. By using perfusion times of varying duration and by studying the entire globe, fluorescein could be traced through the conventional outflow pathway and through an unconventional pathway. The latter consisted of a venous drainage network ending in the vortex veins. The borders of the anterior chamber The nature of the iris anterior border layer has been open to question due to its peculiar structure. Based on the present experiments, and the observations by Vrabec (1952) and by Magari (1957) the physiological importance of the front surface of the iris as a barrier is questioned. Once present in the anterior chamber, fluorescein enters the interstices of the iris stroma, the anterior ciliary body and the trabecular meshwork so rapidly that, as measured by our methods, these anatomical entities form, for practical purposes, a single coextensive fluid compartment. Fluorescein, however, is rigorously excluded from the posterior chamber by the pigmented epithelial barrier at the posterior face of the iris. Fluorescein enters the posterior chamber only via the pupil; actually through the small gap between the iris rim and the front surface of the lens. The conventional ou@ow pathway Since fluorescein was visible in the conventional pathway and absent in the ciliary body in two of the three 5 min experiments, it seems reasonable to suggest that the conventional system f?.lls more rapidly than does the uveal system; this observation has been previously made (Bill, 1967a). A schematic diagram of the conventional pathway for fluorescein is shown in Fig. 11: tracer passes sequentially through the
FIG.
11. Schematic
presentation
of the conventional
outflow
path\%--a!: for anterior
chamber
fluorescein.
trabecular meshwork, Schlemm’s canal, intrascleral channels and thence into episcleral veins. From this point fluorescein passes into larger veins in the extraocular muscles to join the orbital circulation. The leakiness of the veins outside the eye leads to a modest diffusion of tracer into surrounding tissues.
Ii0
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The uveo-vortex outjiou’ ynthwq
The iris stroma in monkeys has been found freely permeable to tracers placed within the anterior chamber (McMaster and Macri, 1968; Saari, 1974; Smelser and Ishikawa, 1962). Jocson and Grant (1965) and Jocson and Sears (1969) have also shown (both in human and monkey eyes) that even a high viscosity silicon polymer injected into the anterior chamber can infiltrate the anterior aspect of the ciliary nrusclc. Further, the penetration of a variety of tracers (indigocarmine (lye, ferritin. thorotrast and inulin) into blood vesselsof the iris and ciliary body has been demonstrated (von Seidel, 1937; Fine, 1964; Ishikawa, 1962; Saari, 1974; Inomata, Bill and Xmelser, 1972; Fowlks and Havener, 1964; Holmberg, 1959; Taniguchi, 1962). while studies using inulin have shown that it passesposteriorly into the suprachoroitl (l&Master and Macri: 1968). Since the opposite is not true, that is. the sameor similar tracers do not leave the blood vesselsof the iris (Von Sallmann and Dillon, 1967; Shakib and Cunha-Vaz, 1966); or of the ciliary muscle (Grayson and Laties, 1971) after intravascular administration. these anterior segment blood vesselsare directIS comparable to retinal blood vessels(C’unha-Vaz and Maurice; 1967). In essenceour report confirms selectedparts of several previous studies. Given the advantage of viewing fluorescein in eyes quick-frozen and then freeze-dried, it is possibleto re-order the aforementioned observations to identify the defining characteristics of a uveo-vortex outflow pathway. The pathwav extends all the way from iris to choroid. Its particulars are as follows : Fluorescein-penetrates blood vesselsin the iris; simultaneously, fluorescein diffusing freely in the interstices of the anterior part of the ciliary muscleenters local blood vessels(Figs 2. 5. i). Thereafter, asthe venous blood flows normally towards the choroicl to leave the eye via vortex veins. tracer from both sources. from iris and from anterior ciliary muscle, mingles together. To the extent that any part of this venous flow leaves the eye through the anterior ciliary complex (for details seeJocson and Grant, 1965 xntl Jocson and Sears. 1969), an intravascular uveo-scleral outflow also ta,kes place. Further, the filling of blood vessels in the ciliary muscle appears to follow a squential pattern: initial general diffusion of the tracer within ciliary muscle tissue is folio\\-ed by k%aliZatioli in connective ti%uC s])acW. and filially, p’I?k’&ti(Jll illtO 1,lood vnW?ls.
In 1964 Fine clearly demonstrated the passageof large moleculesfrom the anterior chamber into the ciliary body. Bill and his collaborators (1965a; 1966; 196ia,b; Inomata. Bill and Smelser (1972)) further demonstrated the passageof large molecules from the anterior chamber into the ciliary body and then into the supraciliary space. From there, in their experiments, the tracers apparently passed out directly through the sclera (Bill, 1965b) and accumulated in the episcleral tissueswhich are drained by conjunctival lymph vessels. The fact that tracers of differing diffusion coefficients movecl along this route at roughly the samerate argued that their passage was due to a bulk fluid flow of acpieoushumor. This route was called the uveo-sclcral pathway, since it invoked both uveal and trans-scleral pathways. Physiological experiments in monkeys showed that this route accounted for about 20% of total outflow at normal intraocular pressure. Flow through the uveo-scleral pathway was found to change little with changesin intraocular pressure,but to be nearly abolished by pilocarpine (Bill, 1967a).
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FLUORESCEIN
In our study fluorescein was observed to leave the anterior chamber primarily by the conventional and uveo-vortex pathways. In all eyes, staining of the sclera mainly ~~~~rred in the immediate vicinity of small, trans-scleral vessels which leaked dye near the limbus. The uveo-scleral pathway and the uveo-vortex pathway, as proposed, are essentially the same within the uvea since, in each, tracer moves from the anterior chamber into the ciliarg body. However, the tracer following the uveo-scleral route is then thought to pass out of the eye through the sclera: whereas that following the uvoovortex route leaves the eye by the vortex veins. Rntmme
of jhorescein
into the weal
venous systenl
The techniques employed do not permit a critical evaluation among competing mechanisms through which fluorescein could enter the uveal blood vessels. Thus without a quantitative measure or the use of inhibitor (Maurice, 19671,our results do not discriminate between the two chief means by which fluorescein could enter the uvoal circulation-active transport or passive ultrafiltration. Fortunately, this pro1A.ln has recently been addressed by Pederson, Gaasterland and XacLellan (1%) who: in comparing the passageof fluorescein to t’hat of lz51-labeledalbumin from t’he anterior chamber into the uveal circulation and vortex veins, found the t1v-o diffcro(l in such a manner to indicate a major role for passivereabsorption of materials. depending on their size. Tracer concentration \~,s measured in a cannulated vortex vein and compared with concentrations in the svst,emic bloodstream. The same
PI,:.
13. Schematic
presentation
of the uveo-vortex
outflow
pathway
for anterior
chamber
fluoreswin.
authors by showing a pressure dependent increase in fluorescein and albumin concentrations in the uveal circulation have furnished a second strand of evidence in favor of a passive ultrafiltration as a mechanism for fluid reabsorption. In these experiments it was found that the contribution of uveo-vortex outflow to total outflow was about lo?;, a value lower than that indicated from previous studies. The anxtSornicalstudies reported here have, therefore, been substantiated by physiological evidence (Pederson et al., 1977). Since as already noted there are strong parallels beta-een the blood vesselsof the retina and iris, the active transport of fluorescein from anterior segment structures is not entirely ruled out (Maurice, 1967). In fact, the participation of sucha mechanism to explain some part of the movement of fluorescein into iris blood vesselsremains an open possibility.
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A. 11. LATIES
In conclusion, we believe there are two separate pathways by which fluorescein leaves the anterior chamber. Iu the conventional pathway (Fig. 11) fluorescein l)as!;es sequentially through the tral)eculaa nleshwork. Schlemm’s ca,nal. t’be aqrueons ant1 cpiscleral veins, the anterior ciliarg veins ant1 hence into the systemic circulation. These vessels often are permeable to t#racer, leading t’o its diffusion into extravascrilar tissues. In the uveo-vortex pathway fluorescein enters the iris and the ciliary muscle directly from the anterior chamber, In the iris, tracer enters blood vessels (Fig. 12). Simultaneously, tracer diffusing posteriorly (Fig. 12) within the ciliary muscle also penetrates blood vessels. Thereafter. a significant amount of fluorescein passes out of the eye via normal nveal drainage through the vortex veins. No general trans.scleral passage of fluoresoein has been found and thus we cannot confirm the existence of a separate extravascular uveo-sclercrl pathway. Since further experiments using fluorescein-labeletl dextran have indicated generally similar patterns of distribution to those found with fluorescein alone, we suggest that even large molecular weight (80 000 daltons) molecules can enter the blood vessels of the iris and ciliary body. Some degree of active transport of free fluorescein into the uveo-vortex pathway camrot be excluded by the present experiments. ACKNOWLEDGMENTS
Supported in part by U.S. Public Health Service Research Grants EY 01413, EY 01329 and EY 01194 from the National Eye Institute. We thank Mm Karen Bowman and Miss Alice McGlinn for their valuable technical assistance, MIX I. Prior for secretarial assistance and Drs J. M. Kling and R. G. Shimp and Mr R. Morrison for their help with animal preparation and anesthesia. We also thank Dr Anders Bill for valuable criticism and for advice on the experiments. REFERENCES 9. (1965a). The protein exchange in the eye with aspects on conventional and uveo-scleral bulk drainage of aqueous humor in primates. In Symposium on Eye Structure, II. (Ed. Rohen, J. W.). Pp. 313-20. Schattauer-Verlag, Stuttgart. Bill, A. (1965b). The aqueous humor drainage mechanism in the cynomolgus monkey (fllacacn irus) with evidence for unconventional routes. Invest. Ophthalmol. 4, 911-9. Bill, A. (1966). Formation and drainage of aqueous humor in cats. Exp. Eye Res. 5, 185-90. Bill, 8. (1967a). Effects of atropine and pilocarpine on aqueous humor dynamics in cynomolgus monkeys. Exp. Eye Res. 6, 120-5. Bill, A. (19’76b). Further st’udies on the influence of the intraocular pressure on aqueous humor dynamics in the cynomolgus monkey. Invest. Ophthalmol. 6, 364-72. Cunha-Vaz, J. G. and Maurice, D. M. (1967). The active transport of fluorescein by the rctinnl vessels and the retina. J. Physiol. (Lord) 191, 467-86. Fine, B. S. (1964). Observations on the drainage angle in man and rhesus monkey: a concept of the pathogen&s of chronic simple glaucoma. Inwest. Ophthnlmol. 3, 609-46. Fowlks, W. L. and Havener, V. R. (1964). Aqueous flow into the perivascular space of the rabbit, ciliary body. Invest Ophthalmol. 3, 374-83. Gersh, I. (1937). Improved histochemical methods for chloride, phosphate, carbonate and potassium applied to skeletal muscles. Anat. Rec. 70, 311-29. Grayson, M. C. and Laties, A. M. (1971). Ocular localization of sodium fluorescein. Arch. Ophthdmol. 85,600-9. Grayson, M., Tsukahara, S. and Laties. A. (1974). Tissue localization in the rabbit and monkey eye of intravenously administered fluorescein. In Fluoresce& Angiography (Ed. Shmizu, K.). Pp. 23546. Igaku Shoin Ltd., Tokyo. Holmberg, A. (1959). The ultrastructure of the capillaries in the ciliary body. Arch. Ophthalmol 62, 94951. Bill,
FATE
OF
ANTERIOR
CHAMBER
FLUORESCEIN
173
Inomata, H., Bill, A. and Smelser, G. K. (1972). Unconventional routes of aqueous humor outflow in cynomolgus monkey (.Macacu irus) Am. J. Ophthalmol. 73, 893-907. Ishikawa, T. (1962). Fine structure of the human ciliary muscle. Invest. Ophthalmol. 1, 587-608. Jocson, V. L. and Grant, W. M. (1965). Interconnections of blood vessels in human eyes. Arch. Ophthalm,ol. 73,707-20. Jocson, V. L. and Sears, M. L. (1969). Ch annels of aqueous outflow and related blood vessels. II. Cercapithecus etbiops, Ethiopian green or green vervet. Arch. Ophthulmd. 81, 24443. Magari, S. (1957). Zur Struktur der Stromas der Iris and zur Frage einea Endothels an Ihrer Vorderflache, Albrecht von Graefes Arch. Ophthalmol. 159, 257-76. Maurice, D. M. (1967). The use of fluorescein in ophthalmological research. Invest. Ophthalmol. 6, 464-77. McMaster, P. R. B. and Macri, F. J. (1968). Secondary aqueous humor outflow pathways in the rabbit, cat and monkey. Arch. Ophthalmol. 79, 297-303. Pederson, J. E., Gaasterland, D. E. and MacLellan, H. M. (1977). Uveo-scleral aqueous outflow in the rhesus monkey: importance of uveal absorption. Ivwest. Ophthalmol. 16, 1008-17. Saari, M. (1974). Morphological basis of diffusion through t,he iris vessels in the dynamics of aqueous humor. Acta. Ophthalm.ol. Suppl. 123,26-M. Shakib, M. and Cunha-Vaz, J. G. (1966). Studies on the permeability of t.he blood-retinal barrier. 1V. Junctional complexes of the retinal vessels and their role in permeability of t#he bloodretinal barrier. Exp. Eye Res. 5,229. Smelser, G. K. and Ishikawa, T. (1962). Investigation on the porosity of the iris. 19th Congress of Ophthalmol. 1. 612-23. Taniguchi, Y. (1962). Fine structure of the blood vessels in the ciliary body. Jap. J. Ophthrtlmol. 6,93-103. Tripathi, R. (‘. (1974). Comparative physiology and anatomy of the outflow pathway. The Eye. Vol. V, (Eds Davson, H. and Graham, L. T., Jr.). Pp. 163-336. Academic Press, N.Y. Von Sallmanrl. L. and Dillon, B. (1967). The effect of diisopropylfluorophosphate on the capillaries of the anterior segment of the eye in rabbits. Am. J. Ophthalm.ol. 30, 1244. von Seidel. E. (1937). Methoden zur untersuchung des intraokularen flussigkeitswechels. In Handbuch der Biologischen Arbeitsmethoden, Abt. V: Methoden zum Studium der Funktionen der einzelnen Organe des tierischen Organismus. P. 1069. Vrabec, F. (19.52). The anterior superficial endothelium of the human iris. Ophfhalmolog%ca. 123, “(l--30.
