Localization of Smooth Muscle Myosin-Containing Cells in the Aqueous Outflow Pathway

Transcription

1 Investigative Ophthalmology & Visual Science, Vol. 31, No. 2, February 1990 Copyright Association for Research in Vision and Ophthalmology Localization of Smooth Muscle Myosin-Containing Cells in the Aqueous Outflow Pathway Annelies W. de Karer,* Sandra J. Spurr-Michaud, and llene K. Gipson Cells containing smooth muscle myosin were localized in the human aqueous outflow pathway by immunohistochemical techniques. In the majority of eyes, immunoreactive cells were observed adjacent to the collector channels and slightly distal to the outer wall of Schlemm's canal. In a few eyes, smooth muscle myosin was localized to cells in the juxtacanalicular tissue and the trabecular meshwork. The immunoreactive cells from these regions may be true smooth muscle cells or pericytes, which can contain smooth muscle myosin. No obvious differences were observed in the pattern of distribution of smooth muscle myosin-containing cells in a comparison of age groups. In the majority of eyes, we observed an apparent direct insertion of the longitudinal portion of the ciliary muscle in the corneoscleral meshwork far internal to the scleral spur. Invest Ophthalmol Vis Sci 31: ,1990 Previous studies have shown the presence of actincontaining smooth muscle-like cells in the human aqueous humor outflow pathway, specifically in the trabecular meshwork and adjacent to the outer wall of Schlemm's canal. 1 ' 2 However, unequivocal evidence that these cells were smooth muscle cells was lacking in these studies, which used actin probes or ultrastructural criteria. Smooth muscle-like cells have been identified in the aqueous humor outflow pathway by ultrastructural methods in the rat 3 and rabbit, 4 " 6 adjacent to the outer wall of Schlemm's canal and at the entrance of the collector channels. In the pig-tailed macaque (Macaca nemestrina), bundles of circumferentially oriented smooth muscle-like cells were detected by transmission and scanning electron microscopy in the outer and posterior part of the trabecular meshwork near Schlemm's canal. 7 Smooth muscle is found in the ciliary body of the human eye, but it has been reported to end at the scleral spur and not to extend into the trabecular meshwork or distal to the collector channels. 8 ' 9 In the From the Eye Research Institute and the *Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts. Supported in part by grant EY (IKG) from the National Eye Institute and by a grant from National Glaucoma Research, a program of the American Health Assistance Foundation. Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida, May Submitted for publication: February 1, 1989; accepted June 7, Reprint requests: Annelies W. de Kater, PhD, Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA human aqueous outflow system, the presence of smooth muscle has not been demonstrated. To determine if the actin-rich cells in the human aqueous outflow pathway 1 ' 2 are in fact smooth muscle cells, we used antibodies specific to smooth muscle myosin for immunohistochemical localization. Materials and Methods Human autopsy eyes (n = 43; ages 1-91 yr), obtained from local Eye Banks and from the National Disease Research Interchange (NDRI), were immersed in a 95% alcohol/5% acetic acid fixative within 24 hr after death. The anterior segments were divided into quadrants and embedded in paraffin by standard procedures. In 36 eyes, at least two quadrants were examined for the presence of smooth muscle myosin. The remaining 7 specimens were obtained as partial segments of the eye, and therefore only one quadrant was examined. Immunofluorescence Seven-micron meridional sections were cut and placed on gelatin-coated slides and dried overnight at 37 C. The sections were deparaffinized in xylene, rehydrated in graded ethanols to phosphate-buffered saline (PBS), ph 7.2, and washed in PBS with 1% bovine serum albumin (BSA) for 10 min. The primary antibody (rabbit antihuman smooth muscle myosin, polyclonal monospecific; specificity previously described 10 " 12 ; a kind gift of Dr. K. Fujiwara, National Cardiovascular Center Research Institutee, Osaka, Japan) was then applied for 1 hr at room temperature in a moist chamber. The slides were rinsed 347

2 348 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Februory 1990 Vol. 31 Table 1. Localization of smooth muscle myosin-containing cells Site of positive binding of antibody Collector channel Outer wall Inner wall Trabecular meshwork Control (ciliary muscle) Collector channels were not observed in 4 specimens. Number of eyes (n = 43) 35/39* with PBS followed by 10 min in PBS with 1% BSA. The secondary antibody (fluorescein isothiocyanate [FITC]-conjugated goat antirabbit IgG; Boehringer Mannheim Biochemicals, Indianapolis, IN) was applied similarly for 1 hr at room temperature. After a PBS wash, coverslips were mounted with a medium of PBS, glycerol, and paraphenylenediamine. 13 Negative control tissue sections (primary antibody omitted) were run routinely with each antibody-binding study. The positive control was the binding of the antibody to the ciliary muscle, which was present in each section. The sections were viewed and photographed with a Zeiss (Oberkochen, West Germany) photomicroscope III equipped for epiillumination. Results In the human aqueous outflow pathway, cells containing smooth muscle myosin were observed adjacent to collector channels, in the pericanalicular region distal to Schlemm's canal, in the juxtacanalicular tissue and within the trabecular meshwork. The pattern of distribution of the immunoreactive cells within a region varied from a single cell to large aggregates. The presence or absence of the cells varied in adjacent quadrants; however, no obvious pattern of binding could be correlated with individual quadrants. In all specimens, positive antibody binding was seen in the ciliary muscle. In negative controls (primary antibody omitted), no binding was present on the ciliary muscle. Table 1 describes the frequency with which we observed the immunofluorescent cells in the aqueous outflow system. Table 2 shows a comparison of positive antibody binding among age groups. A positive correlation between age and the presence of immunoreactive cells was not apparent. Collector Channels In 35 of 39 eyes, discontinuous, single immunoreactive cells were observed directly adjacent to the endothelial abluminal surface of the collector channels. These cells appeared to be oriented parallel to the long axis of the collector channels (Fig. 1). The smooth muscle myosin-containing cells appeared to be elongated fusiform cells with intense specific fluorescence distributed in a bright continuous or intermittent band along the long axis of the cell. Occasionally, smooth muscle myosin-containing cells were observed at or slightly distal to the ostia of a collector channel (Fig. 2). In meridional sections, these immunoreactive cells were positioned either in a circular (Fig. 2a) or longitudinal arrangement (Fig. 2b) with regard to the ostia. Outer Wall In 26 of the 43 eyes, positive antibody binding was seen in cells adjacent to the outer wall of Schlemm's canal. The pattern of binding ranged from large groups of cells exhibiting bright fluorescence (Fig. 3a), to areas of less densely packed cells (Fig. 3b), to single immunoreactive cells (Fig. 3c). The long axis of the smooth muscle-containing cells was always positioned parallel to the longitudinal aspect of the canal. These cells exhibited a pattern offluorescencesimilar to that seen in the immunoreactive cells adjacent to the collector channels. Table 2. Comparison of positive antibody binding among age groups Donor age (yr) Number of eyes (n=.43) Site of positive binding of antibody Ciliary muscle Trabecular meshwork Inner wall Outer wall Collector channel +, binding observed;, no binding observed.

3 No. 2 5MOOTH MUSCLE MYOSIN CELLS IN THE OUTFLOW PATHWAY / de Korer er ol 349 Fig. 1. Immunofluorescence micrographs of collector channels, (a) Discontinuous smooth muscle myosin-containing cells (arrows) adjacent to the lumen of the collector channels (CC). (b) Higher magnification of selected area in (a): a small arteriole with associated smooth muscle cells is present at the lower right of the collector channels. S, sclera. (a, X300; b, X75O). Inner Wall and Trabecular Meshwork In 3 of the 43 eyes, we observed specific antibody binding associated with cells located in the juxtacanalicular tissue adjacent to the inner wall of Schlemm's canal (Fig. 4). In the three specimens showing specific fluorescence, only one or two positive cells were detected. In the trabecular meshwork (corneoscleral or uveal) immunofluorescence was seen in 7 of the 43 eyes. Typically, the smooth muscle myosin-containing cells were localized to the anterior tip of the corneoscleral meshwork at the Umbal margin (Fig. 5). However, positive cells occasionally were seen dispersed throughout the trabecular meshwork. The positive cells in the juxtacanalicular tissue and trabecular meshwork appeared fusiform. The smooth mus- Fig. 2. Smooth muscle myosin immunoreactive cells associated with the ostia of collector channels. In meridional sections, these positive cells (arrows) appear to be oriented in a circular (a) or longitudinal (b) arrangement with regard to the ostia (asterisks). S, sclera; SC, Schlemm's canal; TM, trabecular meshwork. (a, X750; b, X300).

4 350 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1990 Vol. 01 Fig. 3. Micrographs of the aqueous outflow pathway demonstrating three patterns of antibody localization (arrow) adjacent to the outer wall of Schlemrrfs canal (sc). This pattern ranges from a large area of dense localization (a), to several discontinuous cells (b) t to a single positive cell (c). (b, c), sections from different quadrants of the same eye. cm, ciliary muscle; tm, trabecular meshwork, (a, b, c 3 X300). cle myosin in these cells was distributed in a longitudinal pattern, as was observed in the immunoreactive cells associated with the collector channels. Ciliary Muscle Intense fluorescence of the ciliary muscle was observed in every specimen. In many of the specimens, the ciliary muscle appeared to extend through the scleral spur into the deep corneoscleral meshwork (Fig. 6). This anterior extension of the ciliary muscle appeared to be a direct continuation of the longitudinal portion of the ciliary muscle, as suggested by the longitudinal orientation of the smooth muscle myosin-containing cells. Discussion We have demonstrated that in the majority of eyes, smooth muscle myosin-containing cells are found in the human aqueous humor outflow pathway adjacent to collector channels and distal to the outer wall of

5 No. 2 SMOOTH MUSCLE MYOSIN CELLS IN THE OUTFLOW PATHWAY / de Korer er ol 351 Fig. 4. Smooth muscle myosin-containing cell (arrow) in the juxtacanalicular tissue adjacent to the inner wall of Schiemm's canal, s, sclera; tm, trabecular meshwork (X750). Schiemm's canal. We do not know whether these cells are true smooth muscle cells or pericytes, a cell type which also has been shown to contain smoothmuscle-specific myosin. 14 In fact, the positive cells observed adjacent to the collector channels were oriented with regard to the lumen of the channels in a fashion similar to that of the capillary pericytes described by Joyce et al. 15 In both cases, elongated cell bodies are positioned parallel to the long axis of the lumen of the vessel. This position is in contrast to the orientation of smooth muscle cells, which usually encircle the blood vessels. Recently, much evidence of a contractile function of pericytes has been found, 15 " 17 and other observers have suggested that pericytes may participate actively in local blood flow regulation The orientation of the smooth muscle myosincontaining cells observed distal to Schiemm's canal was very similar to the smooth-muscle-like cells that have been identified in the rabbit slightly distal to the angular aqueous plexus, 4 " 6 as well as adjacent to the collector channels in the rat and pig-tailed macaque. 3 ' 7 It is of interest to note that these positive cells did not encircle Schiemm's canal, as smooth muscle cells are observed to do in the vascular system, but appear to be limited to the distal side of the canal. The localization of smooth muscle myosin in the outflow pathway demonstrates the presence of cells adjacent to the collector channels and the outer wall of Schiemm's canal, which may play a significant role in the regulation of aqueous humor outflow. However, further studies are required, to determine whether functional contractility of these cells affects outflow facility. Fig. 5. (a) Micrograph of an immunoreactive cell (arrow) at the anterior tip of the trabecular meshwork (tm). (b) Higher magnification of the positive cell (arrow) demonstrating its fusiform shape, cm, ciliary muscle; s, sclera; sc, Schiemm's canal, (a, X300; b, X750).

6 352 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1990 Vol. 31 Fig. 6. (a) Immunofluorescence micrograph of ciliary muscle (CM) demonstrating its anterior extension (arrow) into the deep corneoscleral meshwork. (b) Negative control: micrograph of same specimen as (a), except that primary antibody was omitted. Note the absence of positive immunofluorescence. S, sclera; SC, Schlemm's canal; TM, trabecular meshwork. (a, b, X300). In the majority of specimens, we observed smooth muscle myosin in the ciliary muscle far internal to the scleral spur. The anterior insertion appeared to be an extension of the longitudinal portion of the ciliary muscle. This observation supports at least in part the conclusions of Kupfer, 20 who studied fetal eyes from 5 months to birth as well as infant and adult eyes to determine the relationship between the meridional muscle of the ciliary body and the corneoscleral meshwork. Kupfer found a continuity between the muscle and the corneoscleral meshwork, and concluded that the majority of the meshwork, except for the juxtacanalicular tissue, represents the tendon of the meridional muscle that inserts into the cornea at Schalbe's line. In a light microscopic analysis of tangential sections, Rohen 21 found three different types

7 No. 2 SMOOTH MUSCLE MYOSIN CELLS IN THE OUTFLOW PATHWAY / de Korer er ol 353 of anterior ciliary tendons, one of which inserts into the scleral spur and one into the corneal stroma. The third one, however, was found to terminate in the juxtacanalicular tissue and corneoscleral meshwork. 22 In an electron microscopy study of tangential sections of normal and glaucomatous eyes, Rohen et al 23 described a network of elasticlikefibers that originate in the ciliary muscle and insert by means of connecting fibrils to the endothelial cells of the inner wall of Schlemm's canal. Hogan et al, 9 however, suggested that there is no extension of the ciliary muscle or its tendons into the peripheral meshwork. Our observations indicate that anterior extensions of ciliary muscle cells insert directly into the deep corneoscleral meshwork internal to the scleral spur. This anterior insertion may contribute significantly to the widening of the intertrabecular spaces during contraction of the ciliary muscle and thus may have a significant influence on outflow facility. In summary, we have demonstrated for the first time that in a majority of human eyes there are cells in the collector channel and outer wall of the Schlemm's canal region that contain smooth muscle myosin. Occasionally, positive cells for smooth muscle myosin were found in the trabecular meshwork and juxtacanalicular tissue. We do not know whether these cells are true muscle cells or pericytes, which also can contain smooth muscle myosin. In either case, these cells appear to contain essential contractile proteins and thus may play a significant role in the regulation of aqueous humor outflow. We have found also that the longitudinal portion of the ciliary muscle appears to insert directly into the deep corneoscleral meshwork far internal to the scleral spur. Key words: aqueous humor outflow pathway, immunohistochemistry, smooth muscle, smooth muscle myosin, pericytes Acknowledgments The authors are grateful to Ellen Zieske for excellent photographic assistance and to Zarina Memon for technical assistance. References 1. Gipson IK and Anderson RA: Actin filaments in cells of human trabecular meshwork and Schlemm's Canal. Invest Ophthalmol Vis Sci 18:547, Grierson I and Rahi AHS: Microfilaments in the cells of the human trabecular meshwork. Br J Ophthalmol 63:3, Tsukahara S: The existence of smooth muscle adjacent to the Schlemm's Canal of the normal albino rat eye. Acta Ophthalmol 56:735, Knepper PA, Farbman Al, and Boondareff W: A smooth muscle plexus associated with the aqueous outflow pathway of the rabbit eye. Anat Rec 182:41, Sakimoto G and Sameshima M: Groups of smooth muscle cells lying close to the intrascleral collector channels located in the anterior chamber angle of the normal albino rabbits: Electron microscopic study I. 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