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Scanning Electron Microscopic Study of the Interior of Schlemm's Canal In the Human Eye
SCANNING ELECTRON MICROSCOPIC STUDY O F T H E INTERIOR O F SCHLEMM'S CANAL IN T H E H U M A N EYE DAVID M. WORTHEN,
M.D.
Gainesville, Florida
Studies in man suggest that the main re sistance to outflow from the anterior cham ber resides betwen the trabecular meshwork and the episcleral veins. Most workers think the major resistance is in the tissues near the inner wall of the canal of Schlemm. The purpose of this series of experiments and observations was twofold: First, we looked for openings on the surface of the inner wall of Schlemm's canal; second, when these were found correlated their frequency with either perfusion pressure or the presence of open-angle glaucoma. Observations on the canal of Schlemm in vervet and rhesus mon keys have been reported by Bill1 and in the marmoset, owl, squirrel, cebus, spider, ver vet, and rhesus monkeys by Anderson. 2 Findings in man have been reported by Spencer, Alvarado, and Hayes, 3 and by Hoffmann and Dumimescu.4 These authors noted openings or indentations on the inner wall of Schlemm's canal of 2 μ or less in di ameter. MATERIALS AND METHODS
Eyes taken at autopsy which were found unsuitable for corneal transplantation formed the bulk of the material for study. The eyes were either immediately fixed in 10% buffered formalin or 4% buffered, osmotically balanced gluteraldehyde ; or, first perfused with physiologic saline solutions as part of a study on outflow dynamics,5 then fixed via the anterior chamber. Seven eyes were perfused with blood, two with Bârâny's solution at pressure of 40 mm Hg and their mates at a pressure of 5 mm From the Research Service, Veterans Adminis tration Hospital, and Department of Ophthalmol ogy, University of Florida College of Medicine, Gainesville, Florida. This investigation was sup ported by a grant from the Veterans Administra tion and from Research to Prevent Blindness, Inc. Reprint requests to D. M. Worthen, M.D., Veter ans Administration Hospital, Gainesville, Florida 32601. 35
Hg. Five eyes were perfused with alpha chymotrypsin and their mates with dilutant. Seventeen eyes were fixed in various ways in a study of preparation techniques. Other eyes were removed at surgery because of the presence of a tumor within the eye. In three cases the eyes were blind and were removed because of pain following trauma. One eye was removed because of absolute glaucoma. In four cases, biopsy specimens were re moved at the time of filtering surgery in cases of open-angle glaucoma. In four cases of congenital glaucoma, biopsy material was studied. Specimens removed at surgery were fixed in gluteraldehyde. Most of the tissue studied was from eyes of patients over the age of 40 years. In general, the eyes were cut in an ante rior-posterior direction, then the segment be hind the pars plana was cut free. Radial cuts were made in the anterior segments resulting in a small sector which was then turned on one side and the excess cornea, sciera, and uvea trimmed. Under the dissecting micro scope, the canal of Schlemm could usually be visualized by retroillumination through the trabecular meshwork. Circumferential cuts were made in the canal in a radial direction resulting in an anterior and a posterior half, each having an inner and outer wall. Various preparation techniques were used. The most satisfactory was freeze-drying. Bits of tissue were placed in 4% buffered osmium tetroxide for one hour. They were sonicated in an ultrasonic cleaner for one second. They were transferred to buffer, then into distilled water for three washings. The tissue was quickly placed into chilled isopentane, then liquid nitrogen. The tissue in liquid nitrogen was placed in a vacuum evaporator either at room temperature or on a chilled platform (Pearse tissue dryer). Vacuum was applied and the tissue was dried for three to 15 hours. When controlled
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JULY, 1972
Fig. 2 (Worthen). Higher magnification of the specimen shown in Figure 1. The bumpy contour of the inner wall contrasts with the smooth surface of the outer wall. The fine filaments crossing the lumen are probably fibrin, as they were not seen in most specimens. The average diameter of the canal was ISO μ with the anterior-posterior diameter being greater than the interior to exterior. 7mJ 7^
cooling was used, the initial temperature was — 65°C. The temperature was gradually raised to room temperature. The tissue, when dried, was placed on double-sided cel lophane tape mounted on aluminium studs. The studs were coated with gold and palla dium and viewed in a Cambridge Stereoscan microscope with original magnifications of 20 to 50,000 powers. Polaroid 55 P / N
film was used to photograph the rasters. RESULTS
In the eyes studied, Schlemm's canal was usually oblong with the long axis parallel to the scierai surface. No difference in the shape was noted as a result of perfusion pressure or the presence of glaucoma. The outer wall tended to be smoother with occa-
Fig. 1 (Worthen). Overview of the angle structures from an eye bank eye of a 72-year-old man with normal intraocular pressures prior to death. Specimen was formalin fixed, freeze-dried. The vertical line indicates the junction of the radial and circumferential cuts. In study of the canal, the circumferential cut would be made posterior to this so as to bisect the canal. The corneal-scleral and uveal trabecular meshwork can be seen to have a different structure in this view, the former being next to the canal with a finer structure and the latter having the wider, more numerous beams. The ciliary muscle is continuous with the uveal meshwork. Above the canal is probably a collector channel going towards the episclera. These were seen leaving the canal in a number of specimens and ranged up to 80 to 100 μ in diameter (XlOO). \\\\\ V
///)) r
Fig. 3 (Worthen). Transmission electron photomicrograph of the inner wall of Schlemm's canal from the biopsy specimen of a 62-year-old man with open-angle glaucoma. Typical of material studied, the nucleus protrudes into the lumen, causing the bumpy contour. A correlation between intracytoplasmic vacuoles and the bumps could not be found.
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Fig. S (Worthen). Inner wall from the normal eye of a 74-year-old man (eye bank tissue, formalin fixed, freeze-dried). This view demonstrates protrusions seen occasionally on the surface of endothelial cells. They range from 0.2 to 0.8 μ in width and from 0.5 to 3.0 μ in length. In this view they can be seen on the center cell but not surrounding ones. They are rarely seen on the outer wall as demonstrated in relief in the upper portion of the photomicrograph (arrow). These protrusions have also been seen in blood vessels in other areas of the eye. \\S\S V 777777^
sional collector channels. I n an overview of the angle area, the corneal-scleral and uveal trabecular meshwork were seen to have a different structure ( F i g . 1 ) . I n a higher power view of the meshwork and canal, there were bumps in the inner wall (Fig. 2) which appear to correspond to nuclei in the endothelial cells ( F i g . 3 ) . N o large channels that might correspond to Sonderman's canals were seen. T h e inner wall had indentations or openings which varied from 0.1 to 1.0 μ in
diameter and had a variable distribution on the cell surface. T h e y usually covered a small amount of surface but in some cases were very numerous ( F i g . 4 ) . T h e openings were noted to cover from 0 . 2 % u p to as much as 8 % of a given endothelial cell sur face. T h e size of the smaller openings is comparable to the openings of micropinocytotic vesicles. Also present on the inner wall were protrusions from the endothelial cell surface which varied from cell to cell ( F i g .
Fig. 4 (Worthen). Enucleated normotensive eye from a 46-year-old woman with a malignant melanoma of the posterior pole. Specimen is formalin fixed, freeze-dried. This view of the inner wall of the canal of Schlemm shows a marked number of openings which were quite variable from one cell to another and from one area of the canal to another. This variation was seen in most specimens studied, but usually not to the degree shown here. No correlation was seen between the large number of openings and the presence of a melanoma in the eye. 77777r
Fig. 6 (Worthen). Inner wall of the canal in a 76-year-old man with known open-angle glaucoma. There are a few openings and two protrusions seen in this view. The presence of either of these struc tures and the presence or absence of glaucoma could not be correlated.
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5). Openings and protrusions were seen in eyes under various conditions of aqueous dy namics (Fig. 6). These protrusions and openings to a greater degree have been seen in blood vessels in other areas in the eye. DISSUSSION
JULY, 1972
confused by cases in which cells, vessels, or other debris block the meshwork. It is the normal-appearing meshwork that is puz zling. Future studies will concentrate on at tempting to quantitate these indentations, to find the relationship of the openings to endo thelial cell vacuoles, and attempts to study ionic and water movement across endothelial cells.
We hoped, in this study, to gain some in sight into the process of aqueous filtration in the normal eye, and that, on this basis, a con SUMMARY trast be drawn between the normal and glauSchlemm's canal was studied by scanning comatous eye. Using scanning electron mi electron microscopy in 50 autopsy eyes, the croscopy alone it is impossible to know if the majority formalin and osmium fixed, freezeopenings are just pits, indentations, or open dried, eye bank tissue. Seven cases of openings into the cytoplasm and/or vacuoles angle glaucoma were included. The inner within the underlying endothelial cells. The wall of the canal was bumpy with protrusion openings were less often located over a nu of nuclei into the lumen. The outer canal was cleus or cell junction, suggesting an associa smoother with clefts, septi, and collector tion with the endothelial cell cytoplasm. channels. Openings or indentations varying However, they varied from one cell to the in size from 0.1 to 1.0 μ were seen consis next, as if they might represent a time-re tently on the inner wall surface. Openings lated structure or a variation in the state of were seen less often if the tissue was aireach cell. Studies to date do not show a dried or prepared by solvent dehydration marked difference between presumed normal methods. The openings varied from one sec and known glaucomatous eyes. Quantitative tion of the canal to another and their pres studies would be required to establish such a ence or absence did not seem to correlate difference. Such a quantitation of these with either the perfusion pressure at the openings is difficult in view of the variation time of fixation or the clinical presence of in their number along the canal wall. The glaucoma. method of tissue preparation provides a key, since air-dried and solvent-dehydrated tissues ACKNOWLEDGMENTS I thank David G. Cogan, M.D., and Toichiro generally do not show the openings as fre quently. Whether these openings or indenta Kuwabara, M.D., for their encouragement and cooperation when this work began in the Howe tions represent a phenomenon concerned Laboratory, Harvard University, Boston, Massa with aqueous filtration is open to question. chusetts; Bruce Ellingson, M.D., and Douglas R. They may only be a consistent artifact or Anderson, M.D., for supplying some of the tissue for study; and Carole Ames for technical assis may represent membrane repair, micropino- tance. cytosis, or other unrelated phenomenon. REFERENCES These may represent a continuing process, 1. Bill, A. : Scanning electron microscopic studies and if a large particle such as a red cell or of the canal of Schlemm. Exp. Eye Res. 10:214, tracer material is present it would be caught 1970. 2. Anderson, D. R. : Scanning electron micros up in the ongoing process. Openings or in copy of primate trabecular meshwork, Am. J. dentations may not relate to the outflow of Ophth. 71:90, 1971. 3. Spencer, W. H., Alvarada, J., and Hayes, F. L. : ionic components and water. It is water and Scanning electron microscopy of human ocular tis ion movement that are of interest in under sues: Trabecular meshwork. Invest. Ophth. 7:651, standing the pathologic process of glaucoma 1968. 4. Hoffmann, F., and Dumimescu, L. : Schlemm's in man. In addition, the small change in their canal under the scanning electron microscope. diameter to account for glaucomatous out Ophth. Res. 2:37, 1971. 5. Ellingson, B. A., and Grant, W. M. : The rela flow resistance might not be detected even if they are a true finding. Clinically, no one is tionship of pressure and aqueous outflow in enucle ated human eyes. Invest. Ophth. 10:430, 1971.
M.D.
Gainesville, Florida
Studies in man suggest that the main re sistance to outflow from the anterior cham ber resides betwen the trabecular meshwork and the episcleral veins. Most workers think the major resistance is in the tissues near the inner wall of the canal of Schlemm. The purpose of this series of experiments and observations was twofold: First, we looked for openings on the surface of the inner wall of Schlemm's canal; second, when these were found correlated their frequency with either perfusion pressure or the presence of open-angle glaucoma. Observations on the canal of Schlemm in vervet and rhesus mon keys have been reported by Bill1 and in the marmoset, owl, squirrel, cebus, spider, ver vet, and rhesus monkeys by Anderson. 2 Findings in man have been reported by Spencer, Alvarado, and Hayes, 3 and by Hoffmann and Dumimescu.4 These authors noted openings or indentations on the inner wall of Schlemm's canal of 2 μ or less in di ameter. MATERIALS AND METHODS
Eyes taken at autopsy which were found unsuitable for corneal transplantation formed the bulk of the material for study. The eyes were either immediately fixed in 10% buffered formalin or 4% buffered, osmotically balanced gluteraldehyde ; or, first perfused with physiologic saline solutions as part of a study on outflow dynamics,5 then fixed via the anterior chamber. Seven eyes were perfused with blood, two with Bârâny's solution at pressure of 40 mm Hg and their mates at a pressure of 5 mm From the Research Service, Veterans Adminis tration Hospital, and Department of Ophthalmol ogy, University of Florida College of Medicine, Gainesville, Florida. This investigation was sup ported by a grant from the Veterans Administra tion and from Research to Prevent Blindness, Inc. Reprint requests to D. M. Worthen, M.D., Veter ans Administration Hospital, Gainesville, Florida 32601. 35
Hg. Five eyes were perfused with alpha chymotrypsin and their mates with dilutant. Seventeen eyes were fixed in various ways in a study of preparation techniques. Other eyes were removed at surgery because of the presence of a tumor within the eye. In three cases the eyes were blind and were removed because of pain following trauma. One eye was removed because of absolute glaucoma. In four cases, biopsy specimens were re moved at the time of filtering surgery in cases of open-angle glaucoma. In four cases of congenital glaucoma, biopsy material was studied. Specimens removed at surgery were fixed in gluteraldehyde. Most of the tissue studied was from eyes of patients over the age of 40 years. In general, the eyes were cut in an ante rior-posterior direction, then the segment be hind the pars plana was cut free. Radial cuts were made in the anterior segments resulting in a small sector which was then turned on one side and the excess cornea, sciera, and uvea trimmed. Under the dissecting micro scope, the canal of Schlemm could usually be visualized by retroillumination through the trabecular meshwork. Circumferential cuts were made in the canal in a radial direction resulting in an anterior and a posterior half, each having an inner and outer wall. Various preparation techniques were used. The most satisfactory was freeze-drying. Bits of tissue were placed in 4% buffered osmium tetroxide for one hour. They were sonicated in an ultrasonic cleaner for one second. They were transferred to buffer, then into distilled water for three washings. The tissue was quickly placed into chilled isopentane, then liquid nitrogen. The tissue in liquid nitrogen was placed in a vacuum evaporator either at room temperature or on a chilled platform (Pearse tissue dryer). Vacuum was applied and the tissue was dried for three to 15 hours. When controlled
36
AMERICAN JOURNAL OF OPHTHALMOLOGY
JULY, 1972
Fig. 2 (Worthen). Higher magnification of the specimen shown in Figure 1. The bumpy contour of the inner wall contrasts with the smooth surface of the outer wall. The fine filaments crossing the lumen are probably fibrin, as they were not seen in most specimens. The average diameter of the canal was ISO μ with the anterior-posterior diameter being greater than the interior to exterior. 7mJ 7^
cooling was used, the initial temperature was — 65°C. The temperature was gradually raised to room temperature. The tissue, when dried, was placed on double-sided cel lophane tape mounted on aluminium studs. The studs were coated with gold and palla dium and viewed in a Cambridge Stereoscan microscope with original magnifications of 20 to 50,000 powers. Polaroid 55 P / N
film was used to photograph the rasters. RESULTS
In the eyes studied, Schlemm's canal was usually oblong with the long axis parallel to the scierai surface. No difference in the shape was noted as a result of perfusion pressure or the presence of glaucoma. The outer wall tended to be smoother with occa-
Fig. 1 (Worthen). Overview of the angle structures from an eye bank eye of a 72-year-old man with normal intraocular pressures prior to death. Specimen was formalin fixed, freeze-dried. The vertical line indicates the junction of the radial and circumferential cuts. In study of the canal, the circumferential cut would be made posterior to this so as to bisect the canal. The corneal-scleral and uveal trabecular meshwork can be seen to have a different structure in this view, the former being next to the canal with a finer structure and the latter having the wider, more numerous beams. The ciliary muscle is continuous with the uveal meshwork. Above the canal is probably a collector channel going towards the episclera. These were seen leaving the canal in a number of specimens and ranged up to 80 to 100 μ in diameter (XlOO). \\\\\ V
///)) r
Fig. 3 (Worthen). Transmission electron photomicrograph of the inner wall of Schlemm's canal from the biopsy specimen of a 62-year-old man with open-angle glaucoma. Typical of material studied, the nucleus protrudes into the lumen, causing the bumpy contour. A correlation between intracytoplasmic vacuoles and the bumps could not be found.
VOL. 74. NO. 1
INTERIOR OF SCHLEMM'S CANAL
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38
AMERICAN JOURNAL OF OPHTHALMOLOGY
JULY, 1972
Fig. S (Worthen). Inner wall from the normal eye of a 74-year-old man (eye bank tissue, formalin fixed, freeze-dried). This view demonstrates protrusions seen occasionally on the surface of endothelial cells. They range from 0.2 to 0.8 μ in width and from 0.5 to 3.0 μ in length. In this view they can be seen on the center cell but not surrounding ones. They are rarely seen on the outer wall as demonstrated in relief in the upper portion of the photomicrograph (arrow). These protrusions have also been seen in blood vessels in other areas of the eye. \\S\S V 777777^
sional collector channels. I n an overview of the angle area, the corneal-scleral and uveal trabecular meshwork were seen to have a different structure ( F i g . 1 ) . I n a higher power view of the meshwork and canal, there were bumps in the inner wall (Fig. 2) which appear to correspond to nuclei in the endothelial cells ( F i g . 3 ) . N o large channels that might correspond to Sonderman's canals were seen. T h e inner wall had indentations or openings which varied from 0.1 to 1.0 μ in
diameter and had a variable distribution on the cell surface. T h e y usually covered a small amount of surface but in some cases were very numerous ( F i g . 4 ) . T h e openings were noted to cover from 0 . 2 % u p to as much as 8 % of a given endothelial cell sur face. T h e size of the smaller openings is comparable to the openings of micropinocytotic vesicles. Also present on the inner wall were protrusions from the endothelial cell surface which varied from cell to cell ( F i g .
Fig. 4 (Worthen). Enucleated normotensive eye from a 46-year-old woman with a malignant melanoma of the posterior pole. Specimen is formalin fixed, freeze-dried. This view of the inner wall of the canal of Schlemm shows a marked number of openings which were quite variable from one cell to another and from one area of the canal to another. This variation was seen in most specimens studied, but usually not to the degree shown here. No correlation was seen between the large number of openings and the presence of a melanoma in the eye. 77777r
Fig. 6 (Worthen). Inner wall of the canal in a 76-year-old man with known open-angle glaucoma. There are a few openings and two protrusions seen in this view. The presence of either of these struc tures and the presence or absence of glaucoma could not be correlated.
VOL. 74, NO. 1
INTERIOR OF SCHLEMM'S CANAL
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AMERICAN JOURNAL OF OPHTHALMOLOGY
5). Openings and protrusions were seen in eyes under various conditions of aqueous dy namics (Fig. 6). These protrusions and openings to a greater degree have been seen in blood vessels in other areas in the eye. DISSUSSION
JULY, 1972
confused by cases in which cells, vessels, or other debris block the meshwork. It is the normal-appearing meshwork that is puz zling. Future studies will concentrate on at tempting to quantitate these indentations, to find the relationship of the openings to endo thelial cell vacuoles, and attempts to study ionic and water movement across endothelial cells.
We hoped, in this study, to gain some in sight into the process of aqueous filtration in the normal eye, and that, on this basis, a con SUMMARY trast be drawn between the normal and glauSchlemm's canal was studied by scanning comatous eye. Using scanning electron mi electron microscopy in 50 autopsy eyes, the croscopy alone it is impossible to know if the majority formalin and osmium fixed, freezeopenings are just pits, indentations, or open dried, eye bank tissue. Seven cases of openings into the cytoplasm and/or vacuoles angle glaucoma were included. The inner within the underlying endothelial cells. The wall of the canal was bumpy with protrusion openings were less often located over a nu of nuclei into the lumen. The outer canal was cleus or cell junction, suggesting an associa smoother with clefts, septi, and collector tion with the endothelial cell cytoplasm. channels. Openings or indentations varying However, they varied from one cell to the in size from 0.1 to 1.0 μ were seen consis next, as if they might represent a time-re tently on the inner wall surface. Openings lated structure or a variation in the state of were seen less often if the tissue was aireach cell. Studies to date do not show a dried or prepared by solvent dehydration marked difference between presumed normal methods. The openings varied from one sec and known glaucomatous eyes. Quantitative tion of the canal to another and their pres studies would be required to establish such a ence or absence did not seem to correlate difference. Such a quantitation of these with either the perfusion pressure at the openings is difficult in view of the variation time of fixation or the clinical presence of in their number along the canal wall. The glaucoma. method of tissue preparation provides a key, since air-dried and solvent-dehydrated tissues ACKNOWLEDGMENTS I thank David G. Cogan, M.D., and Toichiro generally do not show the openings as fre quently. Whether these openings or indenta Kuwabara, M.D., for their encouragement and cooperation when this work began in the Howe tions represent a phenomenon concerned Laboratory, Harvard University, Boston, Massa with aqueous filtration is open to question. chusetts; Bruce Ellingson, M.D., and Douglas R. They may only be a consistent artifact or Anderson, M.D., for supplying some of the tissue for study; and Carole Ames for technical assis may represent membrane repair, micropino- tance. cytosis, or other unrelated phenomenon. REFERENCES These may represent a continuing process, 1. Bill, A. : Scanning electron microscopic studies and if a large particle such as a red cell or of the canal of Schlemm. Exp. Eye Res. 10:214, tracer material is present it would be caught 1970. 2. Anderson, D. R. : Scanning electron micros up in the ongoing process. Openings or in copy of primate trabecular meshwork, Am. J. dentations may not relate to the outflow of Ophth. 71:90, 1971. 3. Spencer, W. H., Alvarada, J., and Hayes, F. L. : ionic components and water. It is water and Scanning electron microscopy of human ocular tis ion movement that are of interest in under sues: Trabecular meshwork. Invest. Ophth. 7:651, standing the pathologic process of glaucoma 1968. 4. Hoffmann, F., and Dumimescu, L. : Schlemm's in man. In addition, the small change in their canal under the scanning electron microscope. diameter to account for glaucomatous out Ophth. Res. 2:37, 1971. 5. Ellingson, B. A., and Grant, W. M. : The rela flow resistance might not be detected even if they are a true finding. Clinically, no one is tionship of pressure and aqueous outflow in enucle ated human eyes. Invest. Ophth. 10:430, 1971.