ACTA OPHTHALMOLOGICA 63 (1985) 493-501

The anatomy of the rabbit aqueous outflow pathway

Jan P. G. Bergmanson

Department of Primary Care (Head: Jerald Strickland). College of Optometry. University of Houston, Texas, USA

Abstract. The morphology of the filtration angle and pathway of aqueous outflow was studied in 10 pigmented rabbits using light and electron microscopic techniques. The following conclusions were arrived at: 1. Numerous pectinate ligaments were found to cross the rabbit filtration angle. However, large spaces between these ligaments allows aqueous access to the peripheral portion of the angle and then to the trabeculum. The drainage of aqueous filtered through the meshwork is served by a plexus of 1 to 4 channels, which contain giant vacuoles along their internal and external walls. 2. A single layer of endothelial cells was found covering all meridians of the entire trabeculum from the termina- tion of the cornea to the filtration angle recess. This layer, here termed the trabecular endothelial layer, is a continuation of the corneal endothelium, and it is ac- companied by the posterior limiting lamina (Descemet’s membrane) for its full length. 3. The aqueous passes through the trabecular endothe- lid layer by an intracellular route that possibly requires energy. 4. The presence of the trabecular endothelial layer in the rabbit may explain the variation that is known to exist between human and rabbit in responses to pharinaceu- tical agents. The physiology of the rabbit trabecular endothelial layer needs to be investigated to determine the suitability of this species as an animal model for the human glaucomatous conditions caused by the presence of such a layer.

Key words: aqueous outflow pathway - trabecular endo- thelial layer - rabbit - electron microscopy.

The rabbit filtration angle is bridged by numerous pectinate ligaments (iris pillars) that project from the iris to the trabeculum (Prince 1964; tripathi 1974). The processes are so numerous that it was

generally held that the peripheral portion of the filtration angle was sealed off by a continuous barrier. The space thus formed was referred to as ‘spaces of Fontana’. However, it has been shown that there are large gaps between the pectinate ligaments giving aquous free access to the ‘spaces of Fontana’ which are, indeed, the peripheral portion of the filtration angle (Prince 1964; Tripathi 1974).

The trabecular region in the rabbit has been shown to differ from that of the primate in some important aspects (Ruskell 1961; Prince 1964; Tri- pathi 1971). Its trabeculum contains fewer meshes than that of the primate and much, if not most of it, faces the portion of the anterior chamber peri- pheral to the pectinate ligaments (Prince 1964), making this region essential to aqueous outflow.

The rabbit eye does not possess a canal of Schlemm, but is provided instead with a plexus of small vessels (Prince 1964; Tripathi 1971). Tripathi ( 1971) terms this plexus the ‘aqueous plexus’ and maintains that they are analogous to the canal of Schlemm in primates. Giant vacuoles are present in the endothelial wall of these vessels and presum- ably have the same function as those found along the internal wall of the canal of Schlemm (Tripathi 197 1).

Prince (1964) and Tripathi (1974) have shown that Descemet’s membrane continues peripherally beyond the limits of the cornea. The presence of corneal endothelial cells around iris pillars was also observed both by Prince (1964) and by Grierson & Chisholm (1978).

The appropriateness of the rabbit as an animal model for human glaucoma has been questioned in earlier studies and physiological experiments have

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indicated some important differences between the rabbit and the primate aqueous outflow facility. For example, Quigley & Addicks (1980) demonstrated that the rabbit eye does not respond like that of the primate to chronically raised IOP. They showed that the rabbit eye will, with increased IOP, undergo a distension, especially of the cornea. There also appears to be an unexplained diffe- rence between the rabbit and the primate in the proportion of aqueous humour drained through the uveoscleral outflow route. According to Bill (1966a,b, 1977), only 2.8% of the aqueous leaves the anterior chamber via this route in the rabbit as compared to 35 % in the monkey. Considering the morphology of the rabbit filtration angle, Tripathi ( 1977) suggested the alternative viewpoint that such a physiological difference between primates and lower placentals did not exist in vivo.

By examining the rabbit outflow pathway, the present study attempts to explain the functional and morphological differences between the fdtra- tion angles of the rabbit and the primate. An

improved understanding of the rabbit aqueous outflow pathway would assist in deciding the future use of the rabbit in glaucoma research.

Methods

Ten pigmented rabbits were examined using go- nioscopy as well as by light and electron micro- scopy. The gonioscopy with a Goldmann 3 mirror lens was performed in vivo after instilling one drop of 0.5 % proparacaine hydrochloride (Alkaine) in each eye.

At a later date, the animals were sacrificed with an intravenous overdose of pentobarbitone sodium (Nembutal). Both eyes were enucleated prior to the cessation of respiratory functions. They were im- mersed immediately in 0.3 % cacodylate-buffered glutaraldehyde and bisected equatorially.

Pieces with the corneoscleral junction and adhe- rent uveal tissue were dissected free at random orientations around the circumference of the tra-

Fig. I . The rabbit filtration angle. The trabecular endothelial layer (E) extends to the angle recess (triangle). A large iris pillar (I) bridging the anterior chamber angle intemipts the endothelial layer. The trabecular meshwork (N) is shallow and

loose. The aqueous plexus (P) is represented by 4 canals. x 250.

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Fig. 2. Cross section through a portion of a rabbit aqueous plexus canal. The lumen (L) of the canal is formed by endothelial

cells, which on the internal trabecular side, are specialized to form giant vacuoles (triangle). x 7500.

beculum. The material amounting to approximate- ly one quarter of the total trabecular circumference was post-fixed for 1 h in 1% osmium tetroxide followed by dehydration through graded alcohol. Thereafter, the tissues were cleared in propylene oxide and embedded in Epon 812. Thin transverse and coronal sections from the preparations of 8 rabbits were obtained in a semi-serial fashion from the whole block, while serial sections were cut over approximately 100 pn from two additional rabbits. The sections were mounted on unfilmed copper grids and stained with 0.5% uranyl acetate in 50% ethanol for 10 min followed by 10 min with 0.4% lead citrate in 0.1 N sodium hydroxide. Some of the preparations, however, were immersed only in the lead citrate. The observations were made with a Jeol JEM lOOC electron microscope. Thicker sec- tions prepared for light microscopy were stained with toluidine blue using the techniques of Trump et al. (1961) and Mayor et al. (1961).

Results

The pectinate ligaments were numerous but show- ed large gaps between them as seen with the gonioscope. It was found in the histological prepa- rations that the rabbit trabecular meshwork was shallow, loose and easily damaged during dissec- tion. At its peripheral and also thickest side, the trabeculum was approximately 10 meshes deep. As in the primate, the rabbit trabecular meshwork contained sheets of endothelial cells which some- times were accompanied by a thick basement mem- brane. Collagen and elastic tissue as well as some melanocytes were found between the endothelial cells forming these sheets. The spaces between the meshes were electron microscopically empty.

The aqueous plexus, found close to and along the external side of the trabeculum, was formed by 1 to 4 vessels (Fig. 1) that have an annular course and in some sections were found to communicate with the venous circulation of the eye.

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The channels forming the aqueous plexus show- ed giant vacuole formations mostly along the inter- nal wall, but sometimes also along the external wall. In places, the wall was very thin and delicate, and the size of the lumen varied considerably from approximately 20 pm to 80 pm (Fig. 2). It was not unusual to find blood cells in the aqueous plexus. The bulk of the trabecular meshwork and most of the aqueous plexus were located peripheral to the pectinate ligaments.

The most striking finding of the present study was the total coverage of the trabecular meshwork by a single layer of endothelial cells which was continuous with the cornea (Figs. 1,3 and 4). These cells were lined peripherally by a thick basement membrane that is known as the posterior limiting lamina or Descemet’s membrane in the cornea. The trabecular endothelial layer stretched all the way from the peripheral cornea to the angle recess and showed minor variation only at junctions with pectinate ligmanents. At such junctions the endo- thelial cells formed a tight but somewhat thinning collar around the iris pillar (Fig. 4). No significant structural modification of the cells forming this layer was observed at its peripheral termination nor was specialization of the adjacent tissue appa- rent at this point. This layer of endothelial cells lining the trabecular meshes was present without interruptions in all the rabbits in all orientations as shown by serial and semi-serial sectioning and, like the other trabecular features, showed no signifi- cant individual differences or meridional varia- tions.

The endothelial cells forming this trabecular layer had a number of mitochondria and pronoun- ced rough endoplasmic reticulum but appeared to contain fewer such organelles than the corneal endothelium (figs. 3 and 4). The thickness of these cells was reduced somewhat peripherally and over the trabeculum the endothelial cells also showed more irregularity in their thickness than was the

case of the corneal endothelium. Zonular junctions such as occludentes were observed between the trabecular endothelial cells (Fig. 3, Inset).

The posterior limiting lamina accompanied the corneal endothelium into the trabeculum to also terminate at the angle recess (Figs. 1, 3 and 4). A tapering of its thickness was noted towards the periphery. Bundles of parallel collagen and elastic fibers were observed at intervals within the poste- rior limiting lamina, and these cords tended to have an annular orientation (Fig. 3).

No smooth muscle cells were found and only a few small nerves were observed in the rabbit tra- beculum (Fig. 5). There was little or no variation between the individual rabbits from any of the above described trabecular components.

Discussion

Although present in large numbers, the pectinate ligaments do not constitute a barrier to aqueous drainage, since spaces were found between them. Consequently, aqueous has free access to this re- gion of the anterior chamber. Tripathi (1977) ar- gues that the spaces peripheral to the pectinate ligament are a ‘peripheral extension of the anterior chamber’. In the present study, it was found that the bulk of the trabecular meshwork is located peripheral to the pectinate ligaments. Therefore, this area in the rabbit is what is known as the filtration angle in the primate rather than a peri- pheral continuation.

Compared to primates, the rabbit trabecular meshwork contains fewer meshes. The present study supports previous observations that aqueous is taken up by a number of small channels (Tripathi 1971; Ruskell 1964) which Tripathi (1971) termed ‘aqueous plexus’. Tripathi & Tripathi (1972) consi- dered the aqueous plexus analogous to the primate

Fig. 3. Trabecular endothelial layer with adjacent trabecular meshes. The endothelial cells (N) forming the meshes are loosely spaced. Similarly, oriented bundles (B) of parallel collagen fibers are positioned between the meshes. Melanocytes (M) and their processes are also observed in this region. Lining the anterior chamber (C) is the trabecular endothelid layer (E) accompanied externally by its basement membrane. Located within this basement membrane is a cord-like structure (triangle), which is formed by a bundle of parallel collagen and elastic fibers. x 4300. Inset: Junction between two trabecular endothelial layer cells. Both endothelial cells contain a large number of rough endoplasmic reticulum (er). Sealing the gap between the cells is a zonula occludens (arrowhead). Within the basement membrane lining these cells is

a bundle of parallel collagen and elastic fibers (triangle). x 8500.

32 ActaOphthal. 63.5 497

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canal of Schlemm, since the channels forming the plexus have giant vacuoles along their internal walls and their function is similar to those of the primate. This conclusion is supported by similar findings in the present study, where giant vacuoles occasionally were also observed along the external wall of the aqueous plexus channels. The frequent observation of blood in the aqueous plexus vessels is in agreement with earlier reports (Ruskell 1964).

Smooth muscle cells other than those associated with the ciliary muscle were not observed. How- ever, scattered smooth muscle cells adjacent to aqueous drainage channels have previously been reported in the rabbit (Knepper et al. 1975; Saki- moto 1979). The functional value of these cells is questionable since they do not appear to be a regular feature around all aqueous channels in the rabbit trabeculum. These reported smooth muscle cells may be ectopic ciliary muscle cells. The pre- sent study is in agreement with Tripathi & Tripathi (1972) who reported only an infrequent occur- rence of nerve fibers in the rabbit trabecular re- gion. The functional significance of the trabecular nerve fibers also seems doubtful in the primate (Ruskell 1976).

The serial and semi-serial sectioning of the fil- tration angle demonstrated the complete coverage of the trabecular meshwork by a layer of endothe- lid cells which is continuous with the posterior cornea. This layer has not been previously report- ed in the rabbit, and the term ‘trabecular endo- thelial layer’ is suggested (Fig. 6). The trabecular endothelial layer stretches to the apex of the angle recess and is accompanied by its basement mem- brane. Previous observations on peripheral projec- tions of the rabbit corneal endothelial cells and posterior limiting lamina did not describe these corneal layers as forming a continuous layer over the trabeculum, nor did they describe how far towards the angle recess they reach (Prince 1964; Tripathi 1974; Grierson & Chisholm 1978). The trabecular endothelial layer was interrupted only

by iris pillars inserting in the trabecular meshes. Since endothelial cells projecting from the trabe- cular endothelial layer formed a tight seal along much of the pillars it is unlikely that a significant proportion of aqueous would escape through such imperfections of the endothelial layer. The zonula occludentes found between the cells in the trabe- cular endothelial layer suggest that the aqueous passes through the cells rather than between them.

Beyond the termination of the trabecular endo- thelial layer at the angle recess, there appeared to be little obstruction to aqueous gaining access to the angular tissue. That a significant proportion of aqueous would seek this route around the trabe- cular endothelial layer to the aqueous channels is unlikely, since these channels are widely separated from the termination of this layer. Indeed some channels forming part of the aqueous plexus were located at the central or anterior extreme of the trabeculum, and it seems more conceivable that the aqueous destined for these channels would flow through the trabecular endothelial layer. Aqueous draining peripheral to the trabecular endothelial layer is more likely to follow the uveoscleral route described by Bill (1966, 1977).

The trabecular endothelial layer is less regular in shape than the central corneal endothelium. Sved- bergh & Bill (1972) noted that a similar loss of regularity is evident in the human and monkey peripheral corneal endothelium. Such surface irre- gularities are probably of little consequence, since this region has no optical function. The basement membrane lining the external side of the trabe- cular endothelial layer is a continuation of the posterior limiting lamina of the cornea and over the trabeculum this basement membrane also lost some of its regularity and thickness. The cord-like bundles of parallel collagen and elastic fibers found within the basement membrane may, together with the pectinate ligaments, have a function in main- taining structural integrity of the fragile rabbit trabeculum. These fibrous bundles may be similar

Fig. 4. Section through the trabecular endothelial layer at the point of insertion of a pectinate ligament. The trabecular endothelial layer (E) projects around the pectinate ligament (I) where its cells are here sectioned flat (triangle). x 6500.

Fig. 5. Trabecular nerve. A small mixed myelinated and unmyelinated nerve (N) is travelling between the trabecular meshes

in a plane parallel to that of the cornea. X 9600.

32* 499

ENOOTHELIAL’/C 9 W

CILIARY BODY r Fig. 6.

Schematic diagram of the rabbit outflow facility showing the trabecular endothelial layer and its relationships.

to those Rohen et al. (1967) reported in what they termed the operculum trabeculi of monkeys. Alt- hough in the rabbit, they did not form as dense a network as was described in the primate.

Rohen et al. ( 1967) described in the cynomolgous and vervet monkeys the trabecular operculum as a peripheral projection of the corneal endothelium and posterior limiting lamina. Only a variable an- terior fraction of the tabecular meshwork was covered by these two corneal layers, which inter- nally were lined by a fibrous membrane.

The apparent conflict between the structure and physiology of the rabbit and primate filtration angles brought out the contradictory results of Tripathi (1977) and Bill (1977), as to the propr- tion of aqueous drained via the uveoscleral path- way. The finding in the present study of a conti- nuous trabecular endothelial layer completely co- vering the trabeculum and the ready access of aqueous to stromal tissue beyond the termination of this layer does not support the finding that only

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2.8 % of the aqueous is drained via the uveoscleral outflow in the rabbit while no less than 35 76 of the aqueous leaves the eye via this route in the monkey (Bill 1977). However, if the relative proportions are indeed correct, it must be concluded that the rabbit trabecular endothelial layer transport aqueous very efficiently from the anterior chamber to the trabe- cular meshes, thus making the uveoscleral outflow an insignificant alternative pathway.

The goniodysgenetic pre-trabecular membranes in congenital glaucomatous patients (Barkan’s membrane) and Chandler’s syndrome are believed to be of endothelial origin (Barkan 1955; Chandler 1956). It is possible that the rabbit trabecular endothelial layer is analogous to these pathological pre-trabecular membranes. However, the existance of such a membrane in the rabbit does not cause an apparent glaucoma. Presumably the rabbit trabe- cular endothelial layer has a different physiology to the human pathological membrane. The presence of pronounced rough endoplasmic reticulum and

many mitochondria in the cells forming the rabbit trabecular endothelial layer points at a metaboli- cally active process for transporting the aqueous from the anterior chamber to the trabeculum. According to Hodson (1977) and Fatt (1978) water is pumped by the corneal endothelium into the anterior chamber by a mechanism involving osmo- larity and pressure differentials. It is possible that the trabecular endothelial layer functions similarly. Further research into the physiology of this stqc- ture in the rabbit is needed to determine if it is a suitable animal model for human glaucomatous conditions caused by the presence of such a layer.

Acknowledgment

I wish to express my gratitude to Professors Donald G. Pitts and Montague Ruben of the University of Houston, University Park, College of Optometry, Houston, Texas, USA and Dr. Bjorn Svedbergh, Department of Ophthal- mology, University Hospital Uppsala, Sweden for offer- ing constructive criticism of the mansucript. Professor Pitts also kindly provided the animals used in the present study. I would also like to thank Dr. Lena W-F Chu for her valuable technical assistance, and Mr. Jay McMichael for his contribution on the artwork.

References

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Received on April 2nd, 1985.

Author’s address:

Dr. Jan P. G. Bergmanson, University of Houston, University Park, College of Optometry, Houston, Texas 77004, USA.

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