Aqueous Humor Pathways Through The Trabecular Meshwork and Into Schlemm'S Canal in the Cynomolgus Monkey (Macaca Irus)

AQUEOUS HUMOR P A T H W A Y S T H R O U G H T H E TRABECULAR MESHWORK AND INTO SCHLEMM'S CANAL IN T H E CYNOMOLGUS MONKEY (MACÀCA IRUS) A N ELECTRON MICROSCOPIC STUDY H A J I M E INOMATA, M.D.,

ANDERS BILL, M.D.,

GEORGE K.

SMELSER, P H . D .

New York, New York

It is generally accepted that aqueous hu­ mor leaves the anterior chamber by a process of bulk flow through the trabecular mesh­ work into the canal of Schlemm. The spaces between the normal uveal and scierai trabeculae are so large that there can be little resis­ tance to flow in this part of the meshwork. The main resistance to flow is thought, rather, to be in the endothelium lining the in­ ner wall of Schlemm's canal or in the endo­ thelial meshwork (endothelial meshwork,1 pore tissue,2 trabeculum cribiforme,3 juxtacanulicular connective tissue 4 ). Although it is accepted that aqueous humor passes through the endothelial meshwork, and that pores exist in the inner wall of Schlemm's canal which permit the passage of particles up to 1-2μ, in diameter,5-15 the actual path­ ways through this area are not quite agreed upon. Many authors have discussed possible channels through the trabecular meshwork, but their studies have been based on light mi­ croscopy only and lacked the resolution nec­ essary to describe the pathways through the pores. So-called Sondermann canals, those relatively wide channels between the intertrabecular spaces and the canal of Schlemm have been regarded by most investigators as This investigation was supported in part by Pub­ lic Health Service Grants S ROI EY 00190-14 and 9 ROI EY 00475-04 from the National Institute of Health; a Grant-in-Aid from the Alfred P. Sloan Foundation; Grant B 71-14X-147-07 from the Swedish Medical Research Council (Dr. Bill) ; a Fight for Sight Postdoctoral Research Fellowship F 210 (G-2). (Dr. Inomata) ; and a Public Health Service Research Career Program Award S-K6NB-19-609-07 from the National Eye Institute (Dr. Smelser). Reprint requests to George K. Smelser, Ph.D., Department of Ophthalmology, Columbia Univer­ sity, 630 West 168th Street, New York, New York 10032.

blindly ending infoldings of the canal of Schlemm. Recently, Iwamoto,16·17 reported finding only a "suggestion" of a connection between such structures and the intertrabecular spaces. Feeney and Wissig,1® who found neither Sondermann channels nor pores through the endothelial cells, suggested that aqueous humor escapes into Schlemm's canal by pinocytosis. A route between or through these cells has been postulated by Fine 19 and by Rohen and van der Zypen.20 It has long been known that large vacuoles ex­ ist in the endothelial cells of the inner wall of Schlemm's canal. Holmberg21·22 was the first to make serial sections of these; he re­ ported that they opened both to the canal of Schlemm and to the endothelial meshwork. He proposed that aqueous humor passed from the spaces in the trabecular meshwork into these vacuoles and, thence, into Schlemm's canal. The possible artifactual nature of these channels has been discussed extensively, since many investigators have been unable to find the pores into the canal of Schlemm.*'18'23 The existence of channels through the endothelial cells has recently been confirmed, however, by several investiga­ tors.24"29 The fraction of vacuoles that have open communications through the endothelial cells is not agreed on. Holmberg seems to con­ sider most, if not all, vacuoles open to both sides, while Kayes 25 has reported that only 1% of the vacuoles communicate with both the intertrabecular spaces and the canal of Schlemm. Tripathi 28 · 27 reports that many vacuoles are wholly intracytoplasmic. The size of the pores on the canal side of the vac­ uoles also was not agreed on. Holmberg21·22 and Bill,30 using transmission and scanning electron microscopy, respectively, found 760

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TABLE 1 D I A M E T E R OF PORES I N T H E ENDOTHELIUM OF SCHLEMM'S CANAL

(ΐΝμ)

Long Pore (Via Vacuole) Species

Investigator

Year

Holmberg21

1959

Human+monkey

0.5-1.5

22

1965 1967

Human -|-monkey Human

1968

Rhesus monkey

0.3-2.0 0.15 and 0.44 0.3-1.5

Tripathi27 1969 Segawa28 1968 Wulle" 1968 0 Bill« 1970 Data in present report

Rhesus monkey Human Human embryo Monkey

Holmberg Kayes25 2

Tripathi «

Canal Side

Minipore

Meshwork Side A little larger than canal side 0.12 and 0.38 A little larger than canal side

0.2-2.0

0.6 0.6

0.3-2.0

Cynomolgus monkey 0.8-1.8

pore sizes between 0.3 and 2μ. Kayes, 25 us­ ing transmission electron miscroscopy, found smaller pores (Table 1). Analysis of the resistance of the aqueous paths through the vacuoles can be made only after the number of functional vacuoles and the distribution of the dimensions of the pores have been further investigated. The re­ sistance to flow through the juxtacanalicular tissue is very difficult to estimate, since the paths between the cells are very irregular and partly filled with fibrils and an unkown substance. We have attempted to determine the actual pathways of aqueous drainage, and to some extent their functional dimen­ sions by introducing particulate material into the anterior chamber of normal monkey eyes without disturbing volume or pressure rela­ tionships, and locating the particles in the trabecular and endothelial meshwork during their passage into Schlemm's canal. The route taken by particles would certainly be available for the aqueous humor leaving the eye. We were concerned mainly with the following questions : 1. Where and how tracer particles pass through the trabecular meshwork into the lu­ men of Schlemm's canal. Do they pass through pores in the vacuoles, between the

Short Pore (Via Flat pore Portion)

0.06

1.0-3.5

0.8-1.8

0.06

endothelial cells, or through Sondermann's channels ? 2. Is there any indication that the pas­ sage of certain sizes of tracers in the range 10 nm to Ιμ. is differentially restricted, i.e., does sieving take place ? 3. If there is sieving, where does it oc­ cur? 4. Are the vacuoles in the inner wall en­ dothelium of Schlemm's canal created intracellularly to open at a certain stage of matu­ rity, or are they always open to one or both sides ? In all previous studies with transmission electron microscopy the eyes were enucleated without control of intraocular pressure, di­ vided and fixed sometimes after rather long periods. It appeared to us that such factors might explain the discrepancies cited by dif­ ferent investigators. We, therefore, fixed the eye in situ immediately after, or even before, death of the animal, and removed the ante­ rior part of the eye without undue rise in intraocular pressure. Initial experiments showed that small rigid particles tended to be lost from the trabecular tissue and the canal when the eyes were processed if they had been injected in a saline medium. Therefore, the perfused particles were suspended in gel-

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atin which was caused to congeal by cooling before the eyes were fixed. MATERIALS AND METHODS

Male and female cynomolgus monkeys (Macaca irus), weighing about 2 kg each, were used. The animals were anesthetized with intravenous sodium pentobarbital, 30 mg/kg. The anterior chambers were cannulated with three needles each introduced through the cornea with a needle gun.32 One cannula was connected to a saline-filled poly­ ethylene tubing, the open end of which was usually set 18 cm above the eye. This served to prevent a rise in intraocular pressure to non-physiologically high levels, and to moni­ tor any variation in intraocular pressure. The other two cannulas connected the ante­ rior chamber by polyethylene tubing to two pushpull coupled syringes.33 About 60μ1 of the particle suspensions were injected into the anterior chamber, but the eye pressure did not change because of simultaneous withdrawal of the same amount of fluid. Continuous mixing of the anterior chamber contents was produced by "jiggling" a small amount of fluid to and fro by means of the push-pull syringes. The tubing monitoring the pressure was clamped 15 minutes after the particle suspension had been introduced into the eye, and the animal was killed by opening the heart to prevent reflux of erythrocytes into Schlemm's canal via the aqueous veins. A minute later the anterior chamber was perfused, by means of the same pushpull syringes, with glutaraldehyde (4% glutaraldehyde in 0.05 M phosphate buffer pH 7.4) which in some cases contained Thorotrast. (Thorotrast was not added to the fixa­ tive when it was the perfused particle). Three minutes later the anterior part of the eye was removed as follows : The external palpebral ligaments were cut, a silk suture was placed through the limbal conjunctiva and, with the suture as a "handle," a keratome incision in the sciera was made several mm posterior to the limbus. This opening was enlarged with scissors until the anterior

MAY, 1972

half of the eye was free. It was then lifted from the orbit, with help of the suture and the perfusion needles, and submerged in cold fixative and the needles then withdrawn. We were satisfied that this method caused the least possible mechanical distortion of the eye, and the anterior chamber was never lost. In those cases in which gelatin had been added to the perfusate, the gelatin was solidi­ fied at autopsy by gently pouring cold water on the eye after killing and after removal of the eyelids. Ten to 15 minutes after killing, the anterior part of the eye was fixed, by im­ mersion only, in glutaraldehyde. In all ex­ periments, the anterior chamber was then opened by radial scissor cuts dividing the an­ terior half of the eye into four quadrants. The blade entered the posterior chamber and the cuts passed through the cornea, sciera, iris, and ciliary body. The lens was cut with a razor blade. The four quadrants were transferred to cold 1% osmium tetroxide in phosphate buffer for 1 hour. After 10 min­ utes in osmium terroxide, the zonula fibers were cut with iris scissors and the lens was removed. The edges of the quadrants were trimmed with sharp razor blades and each was divided in two pieces. The tissues were dehydrated in cold alcohol and were trimmed again in the 80% alcohol stage. The filtration angles appeared open in all cases and, therefore, all reagents could easily reach and diffuse into the trabecular meshwork. Dehydration and treatment with propylene oxide was followed by embedding in Epon 812. The tissue blocks were oriented in two ways : one, to give anteroposterior (meridional) sections of Schlemm's canal (cross sections of the lumen of the canal), the other, a tilted frontal section giving longi­ tudinal sections of the lumen of Schlemm's canal. One-micron-thick sections were stained with Azur II for light microscopy. Serial thin sections, 40-60 nm were cut with a diamond knife on a Porter-Blum ultra mi­ crotome, picked up in good order on singlehole copper grids with a supporting film of parlodion and stained with uranyl acetate

VOL. 73, NO. 5 PATHWAYS THROUGH THE TRABECULAR MESHWORK and lead citrate. The sections on all grids were counted with a dissecting microscope before electron microscopy in order to pre­ vent missing sections. A Siemens Elmiskop 1 was used for the study. PERFUSION MATERIALS AND VEHICLES

ERYTHROCYTES

Blood was taken from a femoral vein by a heparinized syringe and centrifuged. The erythrocytes were washed with an artificial aqueous humor solution34 and resuspended in this at a concentration of about 30%. Two eyes were perfused with leukocytes plus erythrocytes obtained from a buffy coat preparation. The intraocular pressure during perfusion rose to 26-27 cm H 2 0 in these two cases. LATEX SPHERES

These were 0.1, 0.5, and Ι.Ομ in diameter (Dow Chemical). In order to minimize loss of particles, suspensions of 5% latex spheres in 5% gelatin solution were used ; these were obtained by mixing equal volumes of the 10% stock latex sphere suspension with a 10% solution of gelatin. The 10% gelatin was prepared with the artificial aqueous hu­ mor and addition of NaCl to make the final solution isotonic. Use of this concentration of gelatin solution did not elevate the intra­ ocular pressure appreciably, but when twice this concentration was used the intraocular pressure stabilized at 20-22 cm H 2 0 . THOROTRAST

About 0.5 ml of the stock Thorotrast (a 25% stable suspension of thorium dioxide in an aqueous dextran solution ; Testagar & Co.) was added to 10.0 ml of 10% gelatin, producing a flocculent precipitate which was allowed to settle (single particles of the tho­ rium dioxide are about 10 nm in diameter). It was the supernatant fluid, containing an unkown quantity of Thorotrast, which was in­ jected into the anterior chamber. During the next 15 minutes a small amount of anterior chamber fluid passed into the open tubing,

763

preventing a pressure rise to non-physiologic levels. Thorotrast in aqueous media was also used, but results were unsatisfactory due to loss of Thorotrast particles during prepara­ tion. A few experiments were conducted with particles suspended in agar solutions instead of gelatin. These did not penetrate the tra­ becular meshwork as satisfactorily as the gelatin suspensions and were not included in the study. POSTMORTEM

PERFUSION

The anterior chambers of two eyes were perfused with glutaraldehyde and five min­ utes later the animal was killed and the anterior chambers were washed out with modified Ringer's solution. Ten minutes later the anterior chambers were perfused with particles in 5% gelatin in modified Ringer's solution as in the vivo experiments. This perfusion lasted 25 minutes. One eye received a mixture of 0.1 and Ι.Ομ spheres and the other a mixture of 1.0, 0.5, and Ο.ΐμ spheres and Thorotrast. The postmortem eye pressure was maintained at about 12 cm H 2 0 by small injections of fluid from the pushpull syringes. Fixation and processing of the eyes was the same as in other experiments with gelatin. CONTROL EYES

These were simply fixed by perfusion with glutaraldehyde in phosphate buffer. Some eyes were perfused with 40-60μ1 arti­ ficial aqueous humor, followed a minute la­ ter with glutaraldehyde containing Thoro­ trast. OBSERVATIONS

Some attention must be paid to the anat­ omy of the trabecular meshwork and the walls of Schlemm's canal in order to under­ stand the experimental results. ENDOTHELIUM OF SCHLEMM'S CANAL

The endothelial cells of the outer (scierai) wall of Schlemm's canal differ from those of the inner (trabecular) wall in the following

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AMERICAN JOURNAL OF OPHTHALMOLOGY

respects: They rest on a well-defined base­ ment membrane, lack vacuoles and have flat­ tened oval nuclei, and contain no "crystal­ line" inclusions. The basic cytoplasmic organelles, however, are the same in the cells of the outer and inner walls. Endothelial cells lining the inner wall of Schlemm's canal are slender, 60 to 70μ in length, with their long axis parallel to the ca­ nal. Their rounded, but deeply indented nu­ clei are approximately in the center of the cell. Most cells contain vacuoles mainly, but not exclusively, in the perinuclear region. The nucleus and associated vacuoles bulge into the lumen of Schlemm's canal so that the central region of the cell appears to be relatively thick. The portion of the cyto­ plasm on both sides of the nucleus is usually flat and thin, and the cells end in long, rounded tails. The edges of adjacent cells of­ ten overlap. Tight junctions connect the en­ dothelial cells. Cytoplasmic filaments and microtubules run through the cells parallel to their long axes. Small cytoplasmic processes containing filaments extend downward into the endothelial meshwork. Two kinds of pinocytotic vesicles, a larger coated and a smaller, uncoated, are seen. Flocculent mate­ rial is often observed on the surface mem­ brane facing the canal, and in the lumen of vacuoles. Endothelial cells contain mitochon­ dria, rough-surfaced endoplasmic reticulum, glycogen granules, multivesicular bodies, and, rarely, pigment granules and "crystal­ like" inclusions. The latter appear in a regu­ lar lattice pattern, surrounded by a single membrane continuous with the endoplasmic reticulum. The endothelium rests on very fine, unevenly distributed fibrillar material embedded in a homogenous ground sub­ stance, unlike the basement membrane which underlies the endothelium of the outer wall of Schlemm's canal. Most vacuoles range from 1 to 4μ in di­ ameter and are formed by a very thin wall of cytoplasm (Figs. 1 and 2). Some giant vacu­ oles, a few of which are collapsed, are 5 to 8μ in diameter and 13 to 15μ in length. The

MAY, 1972

long axis of these oval vacuoles is oriented parallel to the long axis of the cells. Several vacuoles may be seen in one cell on either side of the nucleus. The number of nuclei and vacuoles in the endothelium lining the inner wall of Schlemm's canal at the light microscopic level were counted. In 110 cross sections of about 200μ of the canal of Schlemm of one preparation, 1300 nuclei and 2461 vacuoles were present. The length of the nucleus in the direction of the length of the canal was about 5-6μ. This means that most nuclei were seen in four sections. The total number of nuclei per mm length of the canal was about 1600. Sizes of the vacu­ oles varied greatly, but there were approxi­ mately 3200 vacuoles in this same region. Vacuoles close to one another may be sep­ arated by only a thin cytoplasmic septum which is often perforated so that they com­ municate with each other. Therefore, vacu­ oles are not necessarily single oval struc­ tures, but may represent interconnecting spaces. All vacuoles open toward the mesh­ work side and some, but not all, are open also to the canal of Schlemm. It is of interest that two openings from one vacuole towards the meshwork side may be found in longitu­ dinal sections. We found only one vacuole with two openings into Schlemm's canal. The diameter of most openings towards the trabecular side was about 2.5μ, with a maxi­ mum of about 3.5μ. The openings from the vacuoles into the canal of Schlemm were smaller, and most pores observed had an ap­ proximate diameter of Ι.Ομ, with a maxi­ mum of about 1.8μ. The length of the pore into the canal of Schlemm and into the tra­ becular spaces was about 0.2-0.4μ (Table 1). Pores were also found in the flat portion of the endothelium—i.e., not in the wall of a vacuole. Complete serial sections from edgeto-edge of these pores (Figs. 3-8) demon­ strate them also to be through a single endo­ thelial cell. Small processes (Figs. 4-8) often made a funnel-like structure leading to the pore and extending into the tissue under­ neath the endothelium. These were rarely

Figs. 1 and 2 (Inomata, Bill, and Smelser). A vacuole in the endothelium (En) lining the inner wall of Schlemm's canal in a control eye (Fig. 1). In an adjacent section (Fig. 2), pores are seen in the vacuole opening both toward the meshwork side and the lumen of Schlemm's canal suggesting a possible route of aqueous humor outflow through these pores. Th: Thorotrast, introduced in the fixative (X 12,000).

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AMERICAN JOURNAL OF OPHTHALMOLOGY

Schlemm's Canal

- Endothelial

MAY, 1972

VOL. 73, NO. 5

PATHWAYS THROUGH THE TRABECULAR MESHWORK

SC

767

■/Λ

Endothelial

Figs. 3-8 (Inomata, Bill, and Smelser). Edge-to-edge serial sections of a pore in the flat, non-vacuolated, portion of the endothelium after perfusion with the monkey's own erythrocytes in the anterior chamber. An erythrocyte (RBC) passes through a pore in the flat portion of the endothelium to enter the lumen of Schlemm's canal (SC). By serial sections, the pore is proven to be definitely intracellular (through one endothelial cell). Note the lines indicating the boundaries of this cell. A funnel-like process of the endo­ thelium is seen to extend toward the meshwork side. Th: Thorotrast ( χ 10,000).

768

AMERICAN JOURNAL OF OPHTHALMOLOGY

MAY, 1972

Schlemm's Canal

Figs. 9-11 (Inomata, Bill, and Smelser). Serial sections through a minipore in a flat portion of the endothelium (En). The minipore, a fenestration 60 nm in diameter with a membranous diaphragm is also formed through a single cell, not between cells. J : Junction. Th: Thorotrast (X 19,000).

VOL. 73, NO. 5 PATHWAYS THROUGH THE TRABECULAR MESHWORK

769

1

se

,

^»τ^*;ϊ'Γ«

Mil*

r^ cn*^ ' " ^τ"^" ^

Fig. 12 (Inomata, Bill, and Smelser). Minipores may also be found in the wall of a vacuole in the endothelium. The size and structure of this minipore is exactly the same as in Figure 10. Dense particles in the lumen of the vacuole are Thorotrast (Th). Sc: Schlemm's canal (x66,000). seen with the pores in vacuoles. Another pore-like structure, Ο.Οόμ. in diameter, but closed by a diaphragm, was observed both in the walls of the vacuoles and in the flat por­ tion of the endothelial cells (Figs. 9-12). This fenestration was also entirely within one endothelial cell. ENDOTHELIAL MESHWORK

The tissue just under the endothelium is different from the corneoscleral meshwork, and is composed of several layers of cells in a matrix of connective tissue components. The fine structure of these cells (Figs. 13-15) differs from that of the cells lining the inner wall of the canal and is much more like those surrounding the trabecular beams. They are randomly disposed in two to five layers and have very long, slender cytoplasmic processes, mostly parallel to the tra­ becular sheets, although some are arranged obliquely or vertically. These touch each other and those of the cells lining Schlemm's canal to make a kind of network. Contacts between these processes are just appositional, without junctional complexes. The nuclei of these cells are oval and their perinuclear cytoplasm much more abundant than

that of the endothelium of Schlemm's canal. Vacuoles and a Golgi complex are often ob­ served in the perinuclear region, and cilia extend into the extracellular space. Cyto-. plasmic filaments, mitochondria, and roughsurfaced endoplasmic reticulum are numer­ ous. Characteristic structures are sacs of a special type of endoplasmic reticulum with many ribosomes arranged in parallel rows (Figs. 13-15). The lumen of this rough sur­ faced endoplasmic reticulum consists of a central clear zone surrounded by dense ho­ mogeneous material. Peripherally, it is con­ tinuous with ordinary rough-surfaced endo­ plasmic reticulum. Pigment granules are lo­ cated in the outer part of the cell around which glycogen granules often accumulate. Pinocytotic vesicles are few, and the "crys­ tal-like" inclusions found in the cells lining the canal are not found. The extracellular matrix consists of fine elastic and collagen fibrils, nerves, and ho­ mogeneous ground substance. A fibrillar ma­ terial is seen just beneath the endothelium lining Schlemm's canal and around the en­ trance to the vacuoles (Fig. 16). Most elastic components 35,3e are located in this area close to the endothelium of Schlemm's canal, and

770

AMERICAN JOURNAL OF OPHTHALMOLOGY

MAY, 1972

Figs. 13-15 (Inomata, Bill, and Smelser). Figure 13 shows a cell in the endothelial meshwork. The endothelial meshwork cells are distinguishable from the endothelium lining the inner wall of Schlemm's canal. They are rich in organelles such as pigment granules, glycogen granules, endoplasmic reticulum, mitochondria and centrioles. They extend long and slender processes which contact the processes of ad­ jacent cells forming a network. The most characteristic structure is a special type of rough-surfaced endoplasmic reticulum. ER: Endoplasmic (χ8,000). In Figures 14 and 15, higher magnification of the special type of endoplasmic reticulum in the endothelial meshwork cells clearly shows the three layers in their lumen : a central clear one and two dense peripheral layers. Ribosomes line up on the outside of the endoplasmic reticulum suggesting active synthesis of some material. The ordinary type of endoplasmic reticulum is seen in continuity with this special type (arrows) (Fig. 14, X23.000; Fig. 15, X26,000).

/Wt r

Figs. 16 and 17 (Inomata, Bill, and Smelser). Figure 16: The endothelial meshwork cell is often located close to a pore in the endothelium (En) of Schlemm's canal (SC). A cytoplasmic process of the endothelial meshwork cell covers the entrance of this vacuole and is accompanied by flocculent and fine fibrillar material ( F ) . E F : Elastic fiber. (χ19,000). Figure 17: A region of the lining of Schlemm's canal showing a firm junction (J) between the endothelial cells and a minipore to the left. An attachment device of the endothe­ lium (En) of Schlemm's canal (SC) to the underlying tissue maintaining its position against the pressure gradient of aqueous humor may be the cytoplasmic process on the meshwork side which contacts a process of the endothelial meshwork cell (arrow). It also clings to the underlying elastic fibers (EF). Note the crystal-like inclusion (Inc) (χ26,000).

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ÄET

SC

771

772

AMERICAN JOURNAL OF OPHTHALMOLOGY

MAY, 1972

are partly surrounded by its cytoplasmic processes (Figs. 9-11 and 17). Collagen fibrils with 64 nm periodicity are distributed in groups within this meshwork, but are less frequent than in the trabecular beams; 100 nm periodicity collagen is rarely observed. A few nerve fibers accompanied by Schwann cells occur in the endothelial meshwork, but no nerve endings were seen.

are seen. In addition, many mitochondria and a great number of aggregated glycogen granules are contained in the nerve fibers, which have a structure characteristic of adrenergic nerves. They do not contact any very specific end organ but instead they terminate in the connective tissue of the trabeculae or near the endothelial cells which cover them.

CORNEOSCLERAL AND UVEAL MESHWORK

PERFUSION EXPERIMENTS

The sceral and uveal meshwork is very similar to that described by Holmberg.21'22 Endothelial cells cover, sometimes incom­ pletely, each trabecular beam, which is com­ posed of a continuous basement membrane, elastic components, collagen fibrils (regular and 100 nm periodicity), nerve elements, and homogeneous ground substance (Fig. 18). The fine structure of the cells covering the trabecular beams resembles closely that of the endothelial meshwork, except that the special rough-surfaced endoplasmic reticulum is seldom seen. The endothelial cells are in contact with those covering adjacent tra­ becular beams by means of cytoplasmic pro­ cesses, so that intertrabecular spaces are sep­ arated into many small channels by a net­ work of these processes. The processes are not joined by junctional complexes but merely contact each other. Nerve fibers, often partly surrounded by Schwann cells, occur in the trabeculae or in the intertrabecular spaces (Figs. 18 and 19). Nerve terminals containing large (80-100 nm), granulated, and small (40-50 nm), clear synaptic vesicles, and with membranes of increased density which may contact the basement membrane surrounding the nerve

It was found that perfused Thorotrast, Ο.ΐμ latex spheres, and blood cells readily passed from the anterior chamber into Schlemm's canal, but fewer 0.5μ and Ι.Ομ spheres entered the canal. Thorotrast, in the gelatin vehicle, penetrated every extracellu­ lar space of the trabecular meshwork except areas with the fine fibrils, and entered each vacuole in the endothelium lining Schlemm's canal (Fig. 20). Thorotrast particles were seen in the pinocytotic vesicles only in cells covering the first or second trabecular beam close to the anterior chamber. Neither Tho­ rotrast nor gelatin was found in pinocytotic vesicles of the endothelium of Schlemm's ca­ nal, nor between adjacent endothelial cells lining the canal. When Thorotrast was per­ fused without a gelatin vehicle, particles were observed to be very unevenly distributed. One-tenth micron diameter latex particles in gelatin were found in the lumen of most vacuoles in the endothelial lining of Schlemm's canal. The concentration of gela­ tin seemed to be the same in the vacuoles as in the canal (Figs. 21-27). However, about 30% of the vacuoles had a lower concentra­ tion of 0.1 μ latex particles in their lumen than was found in the spaces in the juxta-

Figs. 18 and 19 (Inomata, Bill, and Smelser). In Figure 18, a nerve ending is seen in a trabecular sheet of uveal meshwork. The nerve ending contains many small clear vesicles and a few large granulated vesicles (LGV and black arrows). The increased density of its membrane closely apposes basement membrane ma­ terial in the surrounding tissue (white arrow). Endothelium (En) covers the trabecular sheet which is rich in collagen fibers including long spacing collagen fibers (LSGF) (x20,000J. In Figure 19, a nerve ending is seen in a trabecular sheet of uvéal meshwork. Many small clear vesicles (SCV) and large granulated vesicles (LGV) are clearly seen in the nerve ending which also contains aggregated glycogen granules (G) and mitochondria. A Schwann cell partly surrounds the nerve ending ( χ 30,000).

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PATHWAYS THROUGH THE TRABECULAR MESHWORK

773

. ' .

■ ' .VfiP" * j i -

.

1

.4t'

■'· V

".* t . feÎ ' . ; ^

"' ' .

,»·

Figs. 20 and 21 (Inomata, Bill, and Smelser). In Figure 20, taken 15 minutes after perfusion with Thorotrast in 10% gelatin, Thorotrast and gelatin freely enters the intercellular spaces of the endothelial meshwork and into every vacuole (V) of the endothelium. Schlemm's canal (SC) (X4000). In Figure 21, taken IS minutes after perfusion with Ο.ΐμ latex spheres in 5% gelatin, these are seen passing through the pores of the vacuole from the meshwork to the lumen of Schlemm's canal (χόΟΟΟ).

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PATHWAYS THROUGH THE TRABECULAR MESHWORK

canalicular meshwork. At the entrance to these vacuoles, one could sometimes see that particles had been sieved out by the fine fibrillar material (Fig. 28). The Ο.ΐμ parti­ cles also passed through the short pores in the flat parts of the cells. Latex spheres 0.5 to Ι.Ομ in diameter in gelatin reached the endothelial meshwork, but most were trapped by its long cytoplasmic processes and the extracellular sub­ stances, so that a high concentration oc­ curred in the extracellular space in the inner and middle part of the endothelial trabecula (Fig. 29), but relatively few entered the ca­ nal compared to the Ο.ΐμ particles. Perfusion with a mixture of various-sized latex spheres in gelatin demonstrated the sieving process very well. The Ο.ΐμ particles entered the canal, whereas most of the 0.5 and Ι.Ομ particles were held back. A few latex spheres were observed to be phagocytosed by the endothelium covering trabecular beams when these latex spheres were perfused in saline vehicles. Erythrocytes, approximately 6-7μ in di­ ameter, were able to pass through the endo­ thelial trabeculae and enter the vacuoles of the endothelial cells through 2.5-3.5μ diame­ ter pores because they are very plastic and deform readily (Fig. 30). Although in cross sections (to the long axis of the cell) we see one erythrocyte in a vacuole, in longitudinal section from five to seven erythrocytes were often seen in one huge vacuole (Fig. 31). Eythrocytes left the vacuoles through pores 1.0-1.8μ in diameter in the same way that they entered the vacuole. However, since the pores towards the lumen of the canal are smaller and there are fewer than those leading into the vacuoles, they are held back in the vacuoles. Leukocytes used the same route as erythro­ cytes; however, one was seen passing through a pore 3.5μ in diameter (Fig. 32). This was the largest pore into the canal of Schlemm that we observed. The leukocytes, which have a large and possibly more rigid nucleus, may widen the pore during their

775

passage. Before reaching the endothelium of Schlemm's canal, erythrocytes were inhibited in their passage by the cytoplasmic processes of the cells in the endothelial meshwork (Fig. 33), where they accumulated just as did the 0.5 and Ι.Ομ particles. Some erythro­ cytes were seen to go around the ends of the long cytoplasmic processes in order to reach the endothelium lining Schlemm's canal. Erythrocytes were also observed to pass through the short pores Ι.Ομ in diameter in the flat portion of endothelium (Fig. 34). A mixture of three sizes of latex spheres and Thorotrast in gelatin was perfused in an eye 10 minutes postmortem and 15 minutes after perfusion with glutaraldehyde. Most vacuoles in the endothelium were filled with particles of Thorotrast and gelatin, but some contained few particles, and some were al­ most empty. The intercellular spaces of the endothelial meshwork were diffusely filled with Thorotrast particles and gelatin up to the area subjacent to the endothelium of the canal. However, no latex spheres reached the endothelium and entered the vacuoles or Schlemm's canal, since the latex spheres were retained by the cell processes, fibrils, and by the ground substance of the endothelial mesh­ work. When gelatin was perfused through un­ fixed eyes there was swelling of the mito­ chondria and cytoplasm of most cells. It is not clear if this swelling occurred during perfusion, when the gelatin was solidified, or whether it was due to the delayed fixation in these eyes. DISCUSSION

The three-dimensional schematic drawing of the lining of the inner wall of Schlemm's canal and its subjacent tissue summarizes our observations on its anatomy (Fig. 35). We conclude that extracellular pathways ex­ ist through the intertrabecular spaces to the juxtacanalicular tissue which seems to be a special type of loose connective tissue with no well-defined through-and-through routes for fluid passage. Aqueous humor that has

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Figs. 22-27 (Inomata, Bill, and Smelser). Edge-to-edge serial sections of a pore in a vacuole of the endothelium (En) in an experiment with perfusion of Ο.ΐμ latex spheres in 5% gelatin. The pores in the vacuole opening both toward the meshwork and to the lumen of Schlemm's canal (SC) are formed through one endothelial cell ( X 7000).

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777

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VOL. 73, NO. 5 PATHWAYS THROUGH THE TRABECULAR MESHWORK passed the juxtacanalicular tissue can pass through the endothelium of the wall of Schlemm's canal via the pores in vacuoles or through pores in the flat part of the endothe­ lium. There appeared to be no openings be­ tween the endothelial cells of Schlemm's ca­ nal.4·19·20 We found that Sondermann canals, lined by a layer of endothelium, opened to the lumen of Schlemm's canal, but seemed to have no definite direct opening to the trabec­ ular spaces or the anterior chamber. Connections between the intertrabecular spaces and the canal of Schlemm have been demonstrated by several investigators. These are partly filled with fibrils and an unkown substance. This study appears to be the first in which these spaces have been demon­ strated to be functional : We observed a fairly even distribution of gelatin, Thorotrast, and Ο.ΐμ latex particles in the endothe­ lial mesh work, in the vacuoles of the endo­ thelium, and in the pores connecting the vac­ uoles with the trabecular spaces and the lu­ men of Schlemm's canal. There can be no doubt that aqueous humor leaves the anterior chamber through these paths, as suggested by Holmberg.21·22 As pointed out, it was necessary to use gelatin in the perfused fluid to prevent loss of particles during the processing of the eyes. The gelatin did not produce the pores in the cells, however, since these could be seen when fixative only was perfused as in control eyes and in those perfused with erythrocytes. The great deformation of the erythrocytes demonstrated in many of the

779

pictures makes it impossible to conclude much about the dimensions of the pathway through the juxtacanalicular tissue, but this information is provided by the behavior of the latex spheres. T H E ENDOTHELIUM OF SCHLEMM'S CANAL.

The endothelium of the outer wall of Schlemm's canal in Cynomolgus monkeys seems to be very similar to that in m a n

21,22,26,28

The endothelial cells of the inner wall have been reported in previous studies with transmission electron microscopy to be small with a maximum diameter of 8-12μ.21·22 Tripathi 26 reports a maximum length of up to 20μ. A study with scanning electron micros­ copy30 has recently shown that the cells are very long and slender and oriented parallel to the canal. Our results confirm this obser­ vation, as in tilted frontal sections, lengths of 70μ were observed. Experiments with sil­ ver impregnation also show that the cells of the inner wall of Schlemm's canal are long and slender.37 In our sections it was evident that one cell usually contained several vacu­ oles, which often communicate and as a rule were located near the nucleus. A most interesting finding is that pores may exist, not only in the wall of vacuoles, but likewise in the flat portion of endothelial cells. Pores of this type were observed by Segawa28 in the adult eye and by Wulle 31 in an eight-month human embryo. Vegge38 noted, in a human eye, a gap in the flat por­ tion of the endothelium through which a ne-

Figs. 28 and 29 (Inomata, Bill, and Smelser). Figure 28 is 15 minutes after perfusion with Ο.ΐμ latex spheres in 5% gelatin. Although these pass through the pores in most vacuoles, sometimes they are held by the fine fibrillar material (F) at the entrance to some vacuoles which permit only gelatin to enter ( χ 16,000). Figure 29 is another demonstration of the sieving action of the endothelial meshwork IS minutes after perfusion with a mixture of Ο.ΐμ and 0.5,μ latex spheres in 5% gelatin. In contrast to Ο.ΐμ latex spheres, larger particles 0.5μ in diameter hardly reach the endothelium (En) of Schlemm's canal (SC) being retained by the network of cytoplasmic processes of endothelial meshwork cells. The special type of endoplasmic reticulum (ER) is seen in the process of the endothelial meshwork cell. V : Vacuole (X 12,000).

Schlemm's Canal

Vacuole

Figs. 30 and 31 (Inomata, Bill, and Smelser). In Figure 30, an erythrocyte (RBC) is shown entering a vacuole in the endothelium (En) through a pore. It had been perfused into the anterior chamber (X 12,000). In Figure 31, 30 minutes after perfusing the monkey's own erythrocytes in the anterior chamber, a vacuole is seen to be large enough to contain many erythrocytes (RBC) in its lumen. This is a longitudinal section of a vacuole. The erythrocytes had been perfused into the anterior chamber. SC : Schlemm's canal.

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PATHWAYS THROUGH THE TRABECULAR MESHWORK

erotic cell was passing. In the present study, a "mini-pore" 0.06μ in diameter with a thin membranous diaphragm was also often found both in the walls of vacuoles and in the flat portion of the endothelium as well. These may be a stage in the formation of a true pore. Similar diaphragm-closed pores are found in capillaries of many tissues. The sizes of pores in the wall of the vacuoles, thus far de­ scribed, are shown in Table 1. Speakman1·39 measured the pore size in endothelium as 5 to 10μ seen in flat preparations. It would seem that he interpreted the vacuoles as pores. Pores Ι.Ομ in diameter would be almost im­ possible to see by light microscopy. The pore diameter reported in the present work ap­ pears to be a little larger than those in sev­ eral earlier studies using transmission elec­ tron microscopy. This may be because it is impossible to determine their correct size unless complete serial sections are made. The pores are round or slightly oval in shape. It is very interesting that they are almost the same size in the flat portion of the endothe­ lium as in the vacuoles. This may indicate a similarity in origin and function. In order to decide whether the pores of the vacuoles and the flat regions are intra- or intercellular, edge-to-edge serial sections of the pore must be studied. We made many complete serial sections and concluded that all pores were truly intracellular (through the cell) and not, instead, formed by C-shaped cells. Intercellular pathways through the inner wall of Schlemm's canal have been de­ scribed.4'19·20 We could not confirm this. If there is intercellular drainage it must repre­ sent a very small fraction of the total. With scanning electron microscopy, it is not possible to identify the nature of the structures that bulge into the canal of Schlemm from the inner wall. Most of them appeared, however, to be nuclei with associ­ ated vacuoles.80 The results of the present study confirm this conclusion. It is of inter­ est that with the scanning electron micro­ scope it was possible to observe 0.3-2 μ

781

openings into the canal of Schlemm in about 30% of the bulges and the nearby parts of the cells. This leads to the conclusion that only a fraction of the vacuoles are, at any one time, through-and-through channels per­ mitting rapid flow. The existence of a pore through a cell is uncommon. However, the porocyte in sponges, as Ashton 40 has noted, is one exam­ ple. In these species, water passes through minute pores formed in the cytoplasm of the cell, which seem very similar to those re­ ported here. Other, possibly related, phe­ nomena are found in capillary endothelial cells in inflammatory tissue, where lympho­ cytes have been observed to pass through a pore formed in the endothelium.41 In the same kind of inflammation it was found that erythrocytes leak out of growing vessels in the cornea through a pore, proven by serial section to be through the endothelial cell.42 These observations, however, differ some­ what from those concerning the pores in the endothelium of Schlemm's canal, because these were never found empty, but were al­ ways occupied by a cell in transit. In any event, it is clear that a pore can be formed in a cell under certain conditions. Since neither Thorotrast nor gelatin was found in the pinocytotic vesicles of the endo­ thelium, nor in the intercellular spaces be­ tween adjacent cells, it seems unlikely that large volumes of aqueous humor pass this way in preference to the open passageways via vesicles and pores. Feeney and Wissig 18 concluded that vacuoles in the endothelium were not routes of aqueous outflow because of the variation in concentration of ferritin particles within them, as compared to that in the underlying connective tissue. In our ex­ periments perfusion with Thorotrast parti­ cles without the gelatin vehicle did not reveal their true distribution in the tissue during the perfusion, since Thorotrast is easily washed out during fixation. This may also account for the findings of Feeney and Wis­ sig18 and those of Fine 4 · 19 and of Rohen and van der Zypen.20 In our experiments, even

AMERICAN JOURNAL OF OPHTHALMOLOGY

782

MAY, 1972

sçfi .Vacuole WBC

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VOL. 73, NO. 5

PATHWAYS THROUGH THE TRABECULAR MESHWORK

when Thorotrast was added to the fixing fluid, it was recovered in a very irregular way in the chamber angle tissue. Some scat­ tered locations in the trabecular meshwork and occasional vacuoles contained a rather high concentration of Thorotrast, while nearby sites contained none. This means that even the fixative is washed out of the tissue spaces during processing of the eyes. In fact, if washing out of the perfused particles is not prevented, one can expect to find them in the places into which they penetrated rather poorly—and from which they were also lost at a low rate. When trying to understand the formation and function of the vacuoles in the endothelial cells, we must consider the following facts : ( 1 ) not all vacuoles communicate with the canal of Schlemm, (2) all vacuoles seem to be open towards the trabecular side, (3) some vacuoles contain a flocculent material, and (4) 0.1 μ particles did not enter some of the vacuoles within 15 minutes. Taking these facts in consideration, we would like to hy­ pothesize as follows : We start with an endothelial cell which develops an invagination near the nucleus at a place where the attach­ ment to the subjacent tissue is poor. The in­ vagination is partly occluded by the proto­ plasmic processes of the cells in the endothelial trabeculum, and fibrils and ground sub­ stance limit the rate of inflow into the vacu­ ole which is forming. The vacuole enlarges and the wall towards the canal of Schlemm becomes thinner and thinner. Pinocytosis in this wall produces one or several membranecovered minipores. These permit some flow

783

of water, but retain high-molecular-weight substances. The diaphragm in the minipores breaks when the pressure difference between the vacuole and the canal of Schlemm is great enough, and aqueous humor then pas­ ses freely through the vacuole into Schlemm's canal and any flocculent material is washed away. At this stage several new vacuoles may have formed, and these vacuoles tend to open into each other and into the old vacuole. Our results suggest that the endothelial cells may be contractile. We found cytoplasmic filaments and microtubules similar to those in smooth muscle cells arranged par­ allel to the long axis of the cells, and deep indentations of nuclear membrane as seen in contractile vascular endothelium.·43 It is pos­ sible that contraction of the cells contributes to the movement of fluid in and out of the vacuoles which open only towards the trabec­ ular side. In the postmortem eye, after per­ fusion with fixative, the endothelial vacuoles of Schlemm's canal were still open to the lu­ men of the canal and to the meshwork, and both Thorotrast and gelatin passed through some of them into the canalicular lumen. However, compared to the living eye many vacuoles in the postmortem eyes seemed less functional. One hypothesis to explain this phenomenon might be that glutaraldehyde had killed the cells and stopped all contractions, which nor­ mally help to mix the content of the vacuoles with the perfused material. Another hypoth­ esis explaining the observed phenomenon would be that, under normal conditions, dur-

Figs. 32 and 33 (Inomata, Bill, and Smelser). In Figure 32, the anterior chamber had been perfused with the monkey's own leukocytes (a buffy coat suspension). A leukocyte (WBC) is shown just passing through a vacuole in the endothelium through both pores opening toward the trabecular meshwork and to the lumen of Schlemm's canal (SC). A relatively hard nucleus (N) seems to be enlarging the pore. In Figure 33, erythrocytes (RBC) are seen going around the ends of the cytoplasmic processes of endothelial meshwork cells to reach the endothelium of Schlemm's canal. This illustrates their complicated pathway. An erythrocyte is in a vacuole (V) which has a pore opening to the lumen of Schlemm's canal (SC). Th : Thorotrast (X9000).

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Schlemm's Can

MAY, 1972

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PATHWAYS THROUGH THE TRABECULAR MESHWORK

ing a 15-minute period most of the vacuoles had functioned as through-and-through channels for some time, while in the fixed preparations only cells which were open at the moment of fixation permitted flow. This could imply that the pores into the canal of Schlemm possibly open and close quite fre­ quently. A third possible explanation is that fixation created such a high resistance in the juxtacanalicular tissue that at some places gelatin and Thorotrast passed this barrier very poorly. If there is a continuous replacement of old pores by new ones, and eye pressure plays a role in pore formation, it would be easy to understand why many competent investiga­ tors have failed to find the pores in the inner wall endothelium; for unless fixation is rapid, or closing of pores otherwise pre­ vented, few or only small pores, will be visi­ ble. T H E ENDOTHELIAL MESHWORK

The fine structure of the cells forming the endothelial meshwork has not been described in detail as has that of the endothelium covering the trabecular beams.22·23'81'44·45 Although Vegge38 showed that these cells were "very similar" to the endothelial cells of Schlemm's canal, the cells of the endothe­ lial meshwork of the Cynomolgus monkey appear to us as somewhat different from those covering the trabeculae. Asayama46

785

first suggested the importance of this tissue, designating it as different from the trabecu­ lar meshwork proper. The cytoplasmic com­ ponent of the endothelial meshwork, which attracts ones attention immediately, is the highly regular type of rough-surfaced endoplasmic reticulum (Figs. 13-15). It seems to be in an actively synthesizing phase. Fine fibrillar material and a homogeneous sub­ stance are observed around these cells which may be their product and play an important role in the resistance to aqueous outflow. A metachromatic amorphous substance has been demonstrated in the trabecular mesh­ work of human and animal eyes47·48 and, ac­ cording to the investigation of Bârâny and Scotchbrook,49 the resistance to aqueous out­ flow may be reduced by hyaluronidase in the perfusion fluid. Hyaluronidase-sensitive acid mucopolysaccharide, stained with colloidal iron and Alcian blue, was demonstrated in human tra­ becular meshwork by Zimmerman.80 Ac­ cording to Holmberg,21 the region of fine fibrillar material and ground substance coin­ cides with the location of substances having affinity for saccharated iron oxide (mucopolysaccharides). Segawa51 showed the location of these substances by the histochemical col­ loidal iron technique adapted to electron mi­ croscopy. The endothelium lining the inner wall of Schlemm's canal, which lacks a typical base­ ment membrane, must be adherent to the en-

Figs. 34 and 35 (Inomata, Bill, and Smelser). In Figure 34, an erythrocyte is seen passing through a pore in the flat portion of the endothelium (En) to the lumen of Schlemm's canal. The endothelium extends a funnel-like process toward the meshwork side. ER: Endoplasmic reticulum. Th : Thorotrast (X 12,000). Figure 35 is a three-dimensional schematic drawing of the endothelium lining the inner wall of Schlemm's canal and the endothelial meshwork based upon the present study. The endothelial cells of the inner wall of Schlemm's canal are long and spindle-shaped and their nuclear portion, which often contains vacuoles, bulges into the lumen. Vacuoles may occur on both sides of the nucleus. The pores through the endothelial cells are formed in the flat portion of cells as well as in the wall of vacuoles opening both toward the meshwork and to the lumen of Schlemm's canal. The vacuoles and pores are possibly transient structures. The endothelial meshwork cells have long processes which contact those of adjacent cells forming a complicated network. These cells are often located close to the pores in the endothelial cell and are involved in the "sieving" of particles in the aqueous humor. A tiny rectangle in the insert shows the part of the wall of Schlemm's canal that is illustrated.

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AMERICAN JOURNAL OF OPHTHALMOLOGY

dothelial meshwork, otherwise it would col­ lapse into Schlemm's canal due to the higher pressure in the anterior chamber. The nature of the attachment is not quite clear. The cells seem to be adherent to the underlying fine fibrillar material and ground substance. Cytoplasmic processes of these cells extend into the endothelial meshwork, where they attach to the processes of the cells of the mesh­ work, which may serve to anchor it. Some­ times fibrillar material is seen to be interven­ ing between them. The cytoplasmic processes are also observed to cling to the elastic com­ ponents located just beneath the endothelium of Schlemm's canal. This may help the endo­ thelium of Schlemm's canal to maintain its position against the pressure gradient of aqueous humor. The possibility that Schlemm's canal is nor­ mally collapsed and that the trabecular wall rests on, and is supported by, the scierai wall and intervening septa is most unlikely, since there was always at least a narrow space be­ tween the inner and outer walls in our experi­ ments carried out with gelatin perfusion. Interesting structures of the cells in the endothelial meshwork are frequent cilia and centrioles. Vegge23 also reports having ob­ served cilia in the endothelium covering tra­ becular beams. These may serve to regulate aqueous humor outflow by acting as motile organs helping to move mucin through this tissue. The endothelial meshwork is composed of two to five layers of cells with long cyto­ plasmic processes, which produce a very complicated network. In its intercellular meshes there is a net of fine fibrils which is at least partly filled with ground substance material. In living eyes, 0.5 and Ι.Ομ parti­ cles were stopped, to a large extent, in the endothelial meshwork, and even Ο.ΐμ parti­ cles could be seen to pile up in the fibrillar substance at the entrance to some vacuoles. These results suggest very strongly that the functional paths through the juxtacanalicular tissue are narrower than the pores into and out of the vacuoles.52 The aqueous seems

MAY, 1972

to seep through the connective tissue ground substance. The fact that after glutaraldehyde fixation even Ο.ΐμ particles were retained to a very large extent in the juxtacanalicular tis­ sue supports this hypothesis. Fixation creates cross-linking between proteins, and can be expected to have transformed the fibrils into a tight immobile net with rigid meshes.53 If there had been uninterrupted open passage­ ways from the intertrabecular spaces into the canal of Schlemm this would have permitted Ο.ΐμ particles to pass. Since no particles were recovered in the canal of Schlemm in these experiments, it may be concluded that there is no such system of spaces. Our conclusion that the functional pathways through the juxtacanalicular tissue are nar­ rower than those through the endothelial cells in the inner wall of Schlemm's canal does not imply that the resistance in the for­ mer region is higher than that in the latter. The total area filled wi|h fibrils and ground substance, and the length of these paths, must also be considered. Therefore, it ap­ pears that the endothelial meshwork is an important factor affecting the facility of out­ flow owing to the irregularity of the cell bod­ ies, their many protoplasmic processes, and the fibrils and amorphous material contained in the spaces between the cells. All these re­ strict the flow of fluid. The pathology of primary open-angle glau­ coma is not established. Many alterations have been observed in the trabecular meshwork of glaucomatous eyes which may be classed as more or less advanced changes.*'24'54"*0 Loss of the vacuoles in the endothe­ lium of Schlemm's canal has also been re­ ported.24'27 Ashton40 suggested that the ini­ tial obstruction in glaucoma may result from submicroscopic changes or functional abnor­ malities. It would not be surprising if mod­ erate changes in the amount of fibrils and ground substance in the juxtacanalicular tis­ sue might have pronounced effects on the fa­ cility of outflow. We think that perfusion experiments with particles in a suitable ma­ trix in enucleated glaucomatous eyes, with

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PATHWAYS THROUGH T H E TRABECULAR MESHWORK

subsequent electron microscopic analysis, might reveal whether functional pathways through the juxtacanalicular tissue in such eyes are narrower than in normal eyes. It also seems likely that perfusion with parti­ cles in monkey eyes under the influence of cholinergic and adrenergic drugs will help to elucidate the mechanisms through which these substances increase the facility of out­ flow.

787

flexible and that aqueous humor enters the canal of Schlemm almost exclusively through pores in the endothelium of the in­ ner wall of the canal which are either associ­ ated with vacuoles or occur in flat parts of the cells. These pores are all intracellular. Perfusion experiments with particles in a suitable matrix would be most helpful in es­ tablishing the location of the site of in­ creased resistance in enucleated eyes with glaucoma.

SUMMARY

In order to determine the normal outflow pathways of aqueous humor, the contents of the anterior chamber of cynomolgus mon­ keys was mixed with erythrocytes, leuko­ cytes, Thorotrast, 0.1, 0.5, or Ι.Ομ diameter latex spheres, under normal pressure and volume conditions. The blood cells entered the intertrabecular spaces, the endothelial meshwork, and passed into vacuoles in the endothelial cells of the inner wall of Schlemm's canal, and thence to the canal. Pores with a diameter of 0.8-1.8μ in the wall of the vacuoles leading to the canal of Schlemm permitted their passage into the ca­ nal. Experiments with perfusion of Thoro­ trast and latex particles suspended in gelatin, to prevent their loss in preparation, gave re­ producible results which varied with the size of the particles. Gelatin and Thorotrast en­ tered all vacuoles in the endothelium within 15 minutes. Particles Ο.ΐμ in diameter had a lower concentration in 30% of the vacuoles. Pores with a diameter of 0.8-1.8μ were found also in non-vacuolated endothelial cells. Diaphragm closed mini-pores with a di­ ameter of about 60 nm occurred both in the wall of the vacuoles and in endothelial cells. After fixation of the outflow pathways by glutaraldehyde, gelatin and Thorotrast could still pass into and through many vacuoles. The latex particles, however, were all held back in the endothelial trabeculae. The results suggest that the paths through the en­ dothelial trabeculae are very narrow and

REFERENCES

1. Speakman, J. S. : Drainage channels in the trabecular wall of Schlemm's canal. Brit. J. Ophth. 44:513, 1960. 2. Flocks, M.: The anatomy of the trabecular meshwork as seen in tangential section. Arch. Ophth. 56:708, 1956. 3. Rohen, J. W. : Das Auge und seine Hilfsor­ gane. In Handbuk der mikroskopischen Anatomie des Menschen, Bd. 111/2, part 4. Berlin, Springer 1964, p. 285. 4. Fine, B. S. : Observations on the drainage an­ gle in man and rhesus monkey: A concept of the pathogenesis of chronic simple glaucoma. A light and electron microscopic study. Invest. Ophth. 3: 609, 1964. 5. François, J., Neetens, A., and Collette, J. M.: Microradiographic study of the inner wall of Schlemm's canal. Am. J. Ophth. 40:491,1955. 6. Huggert, A.: Pore size in the filtration angle of the eye. Acta Ophth. 33:271, 1955. 7. Huggert, A., Holmberg, A., and Esklund, A. : Further studies concerning pore size in the filtration angle of the eye. Acta Ophth. 33:429, 1955. 8. Huggert, A. : An experiment in determining the pore-size distribution curve to the filtration an­ gle of the eye. Part I. Acta Ophth. 35 :12, 1957. 9. : An experiment in determining the pore-size distribution curve to the filtration angle of the eye. Part II. Acta Ophth. 35:104, 1957. 10. Huggert, A., and Esklund, A. : The location of the chief obstruction to the outflow of aqueous humor from the eye of cattle. Acta Ophth. 36:50, 1958. 11. Peter, P. A., Lyda, W., and Krishna, N. : Anterior chamber perfusion studies. II. Controlled particle size in relationship to pore size. Am. J. Ophth. 44(Pt. 2) :198, 1957. 12. Grant, W. M.: Further sudies on facility of flow through the trabecular meshwork. Arch. Ophth. 60:523, 1958. 13. Karg, S. J., Garron, K. L., Feeney, M. L., and McEwen, W. K. : Perfusion of human eyes with latex microspheres. Arch. Ophth. 61:68, 1959. 14. Hjtfrven, I. : A radioautographic study of erythrocyte résorption from the anterior chamber of the human eye. Acta Ophth. 42:600, 1964. 15. Bill, A. : The elimination of red cells from

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the anterior chamber in vervet monkeys (Cercopithecus ethiops). Invest. Ophth. 7:156, 1968. 16. Iwamoto, T.: Light and electron microscopy of Sondermann's channels in the human trabecular meshwork. von Graefe's Arch. klin. exp. Ophth. 172:197,1967. 17. : Further observation on Sondermann's channels of the human trabecular meshwork. von Graefe's Arch. klin. exp. Ophth. 172:213, 1967. 18. Feeney, L. and Wissig, S. : Outflow studies using an electron dense tracer. Tr. Am. Acad. Ophth. Otolaryng. 70:791, 1966. 19. Fine, B. S.: Structure of the trabecular meshwork and the canal of Schlemm. Tr. Am. Acad. Ophth. Otolaryng. 70:777, 1966. 20. Rohen, J. W. and van der Zypen, E. : The phagocytic activity of the trabecular meshwork endothelium. An electron microscopic study of the vervet (Cercopithecus aethiops). von Graefe's Arch. Klin. exp. Ophth. 175:143, 1968. 21. Holmberg, A.: The fine structure of the in­ ner wall of Schlemm's canal. Arch. Ophth. 62:956, 1959. 22. : Schlemm's canal and the trabecular meshwork. An electron microscopic study of the normal structure in man and monkey (Cercopithe­ cus aethiops). Doc. Ophth. 19:339, 1965. 23. Vegge, T. : Ultrastructure of normal human trabecular endothelium. Acta Ophth. 41:193, 1963. 24. Yamashita, T., and Rosen, D. A.: Electron microscopic study of trabecular meshwork in clini­ cal and experimental glaucoma with anterior cham­ ber hemorrhage. Am. J. Ophth. 60:427, 1965. 25. Kayes, J. : Pore structure of the inner wall of Schlemm's canal. Invest. Ophth. 6:381, 1967. 26. Tripathi, R. C. : Ultrastructure of Schlemm's canal in relation to aqueous outflow. Exp. Eye Res. 7:335, 1968. 27. : Ultrastructure of the trabecular wall of Schlemm's canal (A study of normetensive and chronic simple glaucomatous eyes). Tr. Ophth. Soc. 99:449, 1969. 28. Segawa, K. : Personal communication. 29. : Ultrastructure of Schlemm's canal studied by a replica technique. Jap. J. Ophth. 14:1, 1970. 30. Bill, A. : Scanning electron microscopic stud­ ies of the canal of Schlemm. Exp. Eye Res. 10:214, 1970. 31. Wulle, K. G. : Electron microscopic observa­ tion of the development of Schlemm's canal in the human eye. Tr. Am. Acad. Ophth. Otolaryng. 72: 765, 1968. 32. Sears, M. L. : Miosis and intraocular pressure changes during manometry. Mechanically irritated rabbit eyes studied with improved manometric tech­ nique. Arch. Ophth. 63:707, 1960. 33. Bill, A. and Hellsing, K. : Producation and drainage of aqueous humor in the cynomolgus mon­ key (Macaca irus). Invest. Ophth. 4:920, 1965. 34. Barany, E. H. : Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion. Invest. Ophth. 3:135, 1964.

MAY, 1972

35. Iwamoto, T. : Light and electron microscopy of the presumed elastic components of the trabeculae and scierai spur of the human eye. Invest. Ophth. 3:144, 1964. 36. Yamashita, T., and Rosen, D. A. : The elastic tissue of primate trabecular meshwork. A histologie and electron microscopic study. Invest. Ophth. 3:85, 1964. 37. Liitjen-Drecoll and J. Rohen: Über die endotheliale Auskleidung des Schlemmschen Kanals im Silberimpregnationsbild. von Graefe's Arch. klin. exp. Ophth. 187:247, 1970. 38. Vegge, T. : The fine structure of the trabeculum cribriforme and the inner wall of Schlemm's canal in the normal human eye. Z. Zellforsch. 77: 267, 1967. 39. Speakman, J. : Aqueous outflow channels in the trabecular meshwork in man. Brit. J. Ophth. 43:129, 1959. 40. Ashton, N. : The exit pathway of the aque­ ous. Tr. Ophth. Soc. U.K. 80:397, 1960. 41. Segawa, K., and Smelser, G. K. : Electron microscopy of experimental uveitis. Invest. Ophth. 8:497, 1969. 42. Smelser, G. K.: Recent developments in the histology and histopathology of the cornea by elec­ tron microscop. XXI Concilium Ophth. Acta, 1970. Amsterdam, Excerpta Med., 1971, p. 621. 43. Majno, G., Shea, S. M., and Leventhal, M. : Endothelial contraction induced by histamine-type mediators, an electron microscopic study. J. Cell Biol. 42:647, 1969. 44. Garron, L. K., and Feeney, M. L. : Electron microscopic studies of the human eye. II. Study of the trabeculae by light and electron microscopy. Arch. Ophth. 62:966, 1959. 45. Spelsberg, W. W., and Chapman, G. B. : Fine structure of human trabeculae. Arch. Ophth. 67: 773, 1962. 46. Asayama, J. : Zur Anatomie des Ligamentum Pectinatum. Arch. Ophth. 53:113, 1901. 47. Brini, M. A.: Mise en evidence, a l'aide de techniques histochimiques, d'une substance sensible a l'hyaluranidase dans le trabeculum de l'oeil hu­ main. Bull. Soc. Opht. Franc. 2:256, 1956. 48. Vrabec, F. : The amorphous substance in the trabecular meshwork. Brit. J. Ophth. 41:20, 1957. 49. Bârâny, E. H., and Scotchbrook, S. : Influence of testicular hyaluronidase on the resistance to flow through the angle of the anterior chamber. Acta Physiol. Scand. 30:240, 1953. 50. Zimmerman, L. E. : Demonstration of hyaluronidase-sensitive acid mucopolysaccharide in trabecula and iris in routine paraffin sections of adult human eyes. A preliminary report. Am. J. Ophth. 44:1, 1957. 51. Segawa, K. : Localization of acid mucopolysaccharides in the human trabecular meshwork. J. Clin. Ophth. (Jap.) 24:363, 1970. 52. Barany, E. H. : Pore size and passage of particulate matter through the trabecular meshwork. Doc. Ophth. 13:41, 1959. 53. Bowes, J. H. and Cater, C. W. : The interac­ tion of aldehydes with collagen. Biochem. Biophys. Acta 168:341,1968.

VOL. 73, NO. 5 PATHWAYS THROUGH THE TRABECULAR MESHWORK 54. Teng, C. C, Paton, R. T., and Katzin, H. M : Primary degeneration in the vicinity of the chamber angle. An etiologic factor in wide-angle glaucoma. Am. J. Ophth. 40:619, 1955. 55. Speakman, J. S. and Leeson, T. S. : Site of obstruction to aqueous outflow in chronic simple glaucoma. Brit. J. Ophth. 46:321, 1962. 56. Unger, H. H., and Rohen, J. W. : Biopsy of the trabecular meshwork in 50 cases of chronic primary glaucoma. Am. J. Ophth. 50:37, 1960. 57. Rohen, J. W. and Straub, W.: Elektronen­ mikroskopische Untersuchungen über die Hyalinisierung des Trabeculum corneosclerale beim Sek­

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undärglaukom. von Graefe's Arch. klin. exp. Ophth. 173:21, 1967. 58. Flocks, M.: The pathology of the trabecular meshwork in primary open angle glacuoma. Tr. Am. Acad. Ophth. Otolaryng. 62:556, 1958. 59. Rohen, J. W., and Lütjen, E. : Über die Al­ tersveränderungen des Trabekelwerkes im mensch­ lichen Auge, von Graefe's Arch. klin. exp. Ophth. 175:285, 1968. 60. Valu, L., and Fehér, J. : Altersveränderungen des Trabekel-System, von Graefe's Arch. klin. exp. Ophth. 175:322, 1968.

OPHTHALMIC MINIATURE

In the young days of this king Henry VI, being yet under the gover­ nance of this duke Humphrey, his protector, there came to St. Alban's a certain beggar with his wife, and was walking there about the town beg­ ging five or six days before the king's coming thither, saying that he was born blind, and never saw in his l i f e . . . . When the king was come, and the town full, suddenly this blind man, at St. Alban's shrine, had his sight again.... So happened it then, that duke Humphrey of Gloucester, a man also no less wise than learned, having great joy to see such a mira­ cle, called the poor man unto h i m . . . . He looked well upon his eyes, and asked whether he could see nothing at all in all his life before. And when his wife, as well as himself, affirmed falsely "no," then he looked ad­ visedly upon his eyes again, and said, "I believe you very well, for me thinketh ye cannot see well yet." "Yea sir," quoth he, "I thank God and his holy martyr, I can see now as well as any man." "You can," quoth the duke, "What colour is my gown?" Then anon the beggar told him. "What colour," quoth he, is this man's gown?" He told him also, and so forth : without any sticking he told him the names of all the colours that could be showed him. And when the duke saw that, he bade him be set openly in the stocks . . . for though he could have seen suddenly, by mira­ cle, the difference between divers colours ; yet could he not, by the sight, so suddenly tell the names of all these colours, except he had known them before, no more than the names of all the men, that he should suddenly see. The Acts and Monuments of John Foxe, 1563