Ultrastructure of the Nerves in the Human Trabecular Region

Ultrastructure of the nerves in the human trabecular region

Lynette Feeney

Myelinated nerves were found only in the area of the scleral spur. Unmyelinated nerves were found, in all parts of the trabecular meshwork with the following exceptions: the uveal fibers, the apex of the trabeculae at its juncture with Descemet's membrane, and the outer 7 n which comprise the inner ivall of Schlemm's canal. The nerves travel in the connective tissue of the trabecular sheets, on the endothelial surfaces of the trabeculae, and across the trabecular spaces. They are xorapped by Schwann cell cytoplasm, and, at some point, probably near their termination, become wrapped in endothelial cell cytoplasm. The nerves appear to terminate in folds in the endothelial cells or on the surface of the connective tissue of the trabecular lamellae. The findings are compared ivith the ultrastructure of known sensory and autonomic nerves and nerve endings.

R'oucheron,1 in 1889, described the nerves only passed through the trabeculae nerves of the anterior half of the eye of to end in other tissues. He noted that the the guinea pig, and considered the distribu- area around Schlemm's canal contained tion to be analogous to that in the human many nerves, but no relationship to the eye. Some of the nerve fibers were seen canal itself could be seen. In 1954 Vrabec17 to make a turn around the "pillars" of the studied the innervation of the human trabeculae. "The little organ formed by the trabeculae in 30 to 80 ^ thick tangential nerve fiber winding- in a spiral around a sections stained by a modification of the branch of the pectine pillar seems to merit Gros-Schultze silver technique. The nerve a special name such as corpuscle of trunks were found to arise from the supra- tension." He theorized that as the pressure ciliary nerve plexus and to extend forward in the anterior chamber rose the nerves toward the angle of the anterior chamber lowered it by exertion of a counterpressure where one branch entered the ciliary body on the pillars beneath them. In 1936 stroma and the other entered the trabecu- Kolmer10 described nerves in the tissues of lar meshwork proper. Eleven nerve trunks the anterior chamber angle, but thought the were found to enter the meshwork in a single eye. These fibers divided and assumed a circumferential direction in the trabeculae. He encountered occasional From the Francis I. Proctor Foundation for Re- terminal swellings, but frequently saw the search in Ophthalmology, University of Cali- nerve become thin and vanish beyond the fornia School of Medicine, San Francisco, Calif. resolution of the microscope. Considerable This investigation was supported by Research numbers of nerves were found to pene- Grants B-1229 and B-1782 from the National trate the wall of Schlemm's canal. Holland, Institute of Neurological Diseases and Blind- s ness, United States Public Health Service, von Sallmann, and Collins in 1956 studied Bethesda, Md. the innervation of the human and other 462

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vertebrate anterior chamber angle tissues trabecular endothelial cells, and to make with the Gros-Schultze and the Bodian comparisons with other types of nerves silver impregnation techniques, as well as and nerve terminals. by intravital methylene blue staining of large whole mounts of the eye. In the Methods and materials human being they found that the nerves Electron photographs have been obtained from 11 human eyes for this study. Two normal eyes arise from the supraciliary and ciliary were obtained 15 minutes after death from a 67- plexuses in the region of the scleral spur year-old man. The anterior chambers were per- and terminate as free axonal filaments on fused for 10 minutes with cold buffered osmic the trabecular lamellae and the endothelial acid after which they were opened and placed cells. In the region of Schlemm's canal they in fresh fixative for 3 to 4 hours. found that the nerves penetrate to the The other 9 eyes were obtained after enucleation for malignant melanoma of the choroid. Four endothelium of the canal and end at the of these were perfused as noted above. The other base of the cell, or are separated from the 5 were opened immediately and placed in cold canal by several layers of endothelial cells. 1 per cent osmic acid buffered to 7.4 with Studies of degeneration followed this re- barbital buffer. The surgical specimens were port.0 The authors concluded that the nerve fixed within 3 minutes of enucleation for 3 to 4 hours. One millimeter strips of tissue were cut to fibers of the trabecular meshwork are de- include peripheral cornea, trabecular meshwork, rived from at least three sources, namely, and the corona of the ciliary body. The tissues the parasympathetic, sympathetic, and were dehydrated in ethanol and embedded in an fifth nerves. 8:2 mixture of n-butyl and methyl methacrylate. 11 Tangential and meridional sections were cut on Kurus in 1958 demonstrated nerves in the Porter-Blum or LKB ultratome, and mounted the trabecular meshwork of the human on Formvar or carbon-coated copper grids. Some eye by the silver impregnation method of sections were stained with saturated uranyl Gros. He also showed degenerative changes acetate, 1 per cent phosphotungstic acid, or lead in the nerve fibers of the trabeculae of hydroxide. All sections were viewed in the RCA the aged and that these changes corre- EMU 3E electron microscope. sponded with those seen in preglaucoma Results states. Both myelinated and unmyelinated Wolter20 in 1959 demonstrated trabecu- nerves were found in the trabecular mesh- lar nerves in the eye of a 2-month-old in- work of human eyes. fant by the use of the del Rio Hortega Myelinated nerves were observed only silver carbonate stain. Many of the nerves in the region of the scleral spur, entering merely passed through the trabeculae to from both the ciliary muscle and the end in the comeal endothelium. Others scleral regions. Myelination of the nerves terminated in the trabecular endothelium. varied in different eyes and in different The author later reported degeneration of portions of the same eye. The myelinated the nerve fibers in a case of open-angle nerves were about 3 ^ in diameter, with glaucoma in a human being. numerous Schwann cell wrappings forming Recent interest in the nerves of the a myelin collar about 0.5 p, in thickness trabecular meshwork suggested a study of around the nerve. the ultrastructure of these nerves. Numer- The axon of the nerve was seen in the ous reports have appeared describing the center of the myelin sheath separated from ultrastructure of nerves, nerve endings, the sheath by an intercellular space be- their relationships to Schwann and sup- tween the plasma membrane of the axon, porting cells, sensory receptor cells, and or axolemma, and that of the myelin motor cells. The purpose of this paper is sheath. The axoplasm was filled with fine to describe the fine structure of human neurofilaments, dense slender mitochondria, trabecular nerves, their relationship to the and occasional elements of the endoplasmic Schwann cell, to the trabeculae and reticulum (Fig. 1).

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Figs. 1 and 2. For legends see page 469.

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ENDO

SCH

Figs. 3 and 4. For legends see page 469.

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Figs. 5 and 6. For legends see page 469.

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Fig. 7. For legend see page 469.

ENDO 1

SV M SV

TS ENOO

Fig. 8, A and B. For legend see page 469.

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10 Figs. 9 and 10. For legends see page 469.

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Fig. 1. Electron micrograph of a myelinated nerve fiber in the trabecular meshwork near the scleral spur. TS, Trabecular space. CC, Material with 1,000 A banding. MY, Myelin. NF, Nerve fiber. SN, Schwann cell nucleus. (x 10,000.)

Fig. 2. Electron micrograph of an unmyelinated branching nerve trunk in the meshwork near the scleral spur. One branch is seen to the left projecting from a nodular swelling. The second branch occurred in a slightly different plane. A layer of Schwann cell cytoplasm (SCH) envelopes the nerve fiber (NF). Cytoplasm of a trabecular endothelial cell is seen at ENDO. (xll,000.)

Fig. 3. Electron micrograph of an unmyelinated nerve fiber (NF) and its Schwann sheath (SCH) in the connective tissue of a trabecular lamella. This is a flat section through the layer of ground substance which contains the 1,000 A material (CC). ENDO, Trabecular endothelial cell. (x8,000.)

Fig. 4. Electron micrograph of a nerve fiber (NF) en route through a trabecular space (TS). The axoplasm of the nerve fiber contains slender dense mitochondria (M) and fine neurofilaments. An intercellular space of about 150 A (arrows) separates the nerve fiber from the Schwann cell cytoplasm (SCH). Cross sections of the collagen cores of 2 trabecular lamellae are seen at C and C. ENDO, Trabecular endothelial cell. (x35,000.)

Fig. 5. Electron micrograph of a portion of a Schwann cell and several nerve fibers. The Schwann cell nucleus (SN) occupies the right half of the picture, its cytoplasm extends to the left, and contains over a dozen small nerve fibers (NF) in deep or shallow pockets. Each nerve is separated from the Schwann cell by an intercellular space of about 150 A (arrows) and is connected to the surface by a mesaxon (MES). Several of the outermost nerve fibers are incompletely covered by Schwann cell neurolemma, and two of them (A,B) contain numerous vesicles with light centers similar to those seen at synapses. (x34,000.)

Fig. 6. Electron micrograph of a nerve fiber becoming enveloped in endothelial cell cytoplasm. Two trabecular lamellae (TL) are seen and both are covered by endothelial cell cytoplasm (ENDO). The nerve fiber (NF) is partially covered by cytoplasm from the Schwann cell the nucleus of which is seen at SN. Arrows mark the termination of the Schwann cell cytoplasm. Endothelial cell cytoplasm sheaths the nerve below the arrows. (xl0,000.)

Fig. 7. Electron micrograph of a nerve fiber wrapped by cytoplasm of a trabecular endothelial cell. The collagen core (C) of a flat sectioned trabecular sheet (TL) appears at upper left. The endothelial covering (ENDO) of the trabecular sheet encloses a nerve fiber (NF) of which the mesaxon-like connections (MES) to the cell surface indicate the extracellular course of the nerve through the endothelial cell. (xl2,000.)

Fig. 8. Electron micrograph of nerves showing the elements typical of terminal axoplasm. A, Numerous vesicles (SV) and portions of two mitochondria (M) are visible. Cytoplasm of an endothelial cell (ENDO) surrounds the nerve. Two dense lines limit the nerve (arrows): the inner line is the plasma membrane of the nerve, the outer one is the plasma membrane of the trabecular endothelial cell. TS, Trabecular space. (x22,000.) B, Similar to 8A. C, 1,000 A material in a trabecular lamellae. (x22,000.)

Fig. 9. Electron micrograph of trabecular nerve which appears to end on the connective tissue of a trabecular lamella. A portion of the nucleus (N) of an endothelial cell is seen. The nerve fiber (NF) passes very close to this nucleus. Double arrows mark the closely apposed plasma membranes of the nerve and the endothelial cell. C, Collagen of the trabecular lamella. M, Mitochondria. ENDO, Endothelial cell cytoplasm. (xl8,000.)

Fig. 10. Electron micrograph of nerve fibers which contain numerous vesicles (V) and larger dense bodies (B) in the neuroplasm. ENDO, Endothelium of the trabecular sheet (TL). CC, Material with 1,000 A banding. (x22,000.)

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The transition from complete myelina- sheets. There may be confusion regarding tion to lack of myelination was abrupt the neurolemmal cell of those nerves since few intermediate forms of myelina- traveling in the trabecular spaces and on tion were found. Occasional nerves were the surface of the trabeculae. However, it seen wrapped only twice by the Schwann was assumed that the cytoplasm most cells. The membranes, however, were not closely applied to the nerve was that be- compressed into the typical dense myelin longing to the Schwann cell. Small nerves layers, and, presumably, they represented may be incompletely covered by the nerves close to the termination of the Schwann cell. In other sections several myelin sheath. small nerves shared the same neurolemma Unmyelinated nerve fibers were found (Fig. 5). in all parts of the trabecular meshwork Occasionally, nerves were found to with the following exceptions: the uveal emerge from a trabecular sheet and be- meshwork, the apex of the meshwork near come invested with a sheath of endo- its insertion into Descemet's membrane at thelial cell cytoplasm. The endothelial cell Schwalbe's line, and in the outer 7 ^ of the was seen covering the surface of the meshwork which comprised the inner wall trabecular sheet and at the same time en- of Schlemm's canal. closing a nerve fiber in a pocket or closed One hundred nerves averaged 0.9 p in fold in its cytoplasm (Fig. 6). The endo- diameter with a size range of 0.2 to 3.0 f*. thelial cell could be distinguished from The largest nerves were seen in the mesh- the Schwann cell because of a much larger work near the scleral spur. In flat sections nucleus, large saccules of endoplasmic they branched dichotomously, the fork reticulum in the cytoplasm, and an exten- of the branch usually marked by a nodular sive Golgi apparatus. Nerves were seen swelling (Fig. 2). The smallest nerves which deeply invaginated the endothelial were found in the midtrabecular zone at cell and indented the nucleus. Although a level with the anterior limit of the canal these nerves had an apparent intracellular of Schlemm. No nerves were seen in the course in the endothelial cell (suggested portion of the trabeculae in which it began by light microscopy), the actual extra- to merge with corneal tissue. Many nerves cellular relationship could be demon- were found in the trabeculae 20 to 30 /* strated. The deeply infolded plasma mem- from the canal, and large bundles of both brane of the endothelial cell, comparable myelinated and unmyelinated nerves were to the mesaxon of the Schwann cell wrap- found as close as 20 /A in the scleral tissue ping, could be traced to the surface of the external to the canal. cell at the point of entrance (Fig. 7). Thus, Some unmyelinated nerves extended an intercellular space of about 150 A sepa- into the meshwork via the connective tis- rated the nerve from the trabecular endo- sue of the trabeculae. They lie in the thelial cell. ground substance among the islands of The fine structure of the nerve axon 1,000 A-banded material close to the col- was comparable to that of peripheral nerves lagen core (Fig. 3). Others lie on the seen in other tissues. The axoplasm of the free surface of the trabeculae, closely nerve contained numerous, dense, solid applied to the endothelial cells. The nerves neurofilaments 100 to 200 A in diameter. frequently passed through the trabecular The mitochondria were long and slender spaces from one lamella to another. and had a strikingly dense matrix in which There was little doubt that the Schwann the cristae generally ran longitudinally or cells formed the neurolemmal covering of obliquely. Granular endoplasmic reticulum the nerves in the region of the scleral spur was seldom seen, but agranular vesicles and ciliary muscle, and those traveling in were frequently an outstanding feature the connective tissue of the trabecular of the axoplasm.

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Nerve endings. Structures closely re- The nerves have a common feature: They sembling nerve endings were seen either in all lose their Schwann cell covering near the trabecular endothelial cells (Fig. 8) or the terminus and either become wrapped at times on the connective tissue surface of in the cytoplasm of a local cell or continue a trabecular lamella (Fig. 9). The axon be- as bare axons (organ of Corti). Other came slightly larger, forming a bulb 1 to sensory nerve endings, such as those of the 2 fj, in diameter and the axoplasm contained muscle spindle1*1 and digital tactile cor- an unusually large number of mitochondria. puscles,13 retain the Schwann sheath Vesicles of the size associated with synaptic throughout their course. axoplasm generally were present, without The question arises whether the electron preferential accumulation at the plasma microscope has provided any morphologic membrane. Occasional dense bodies about evidence which will distinguish nerve end- 0.1 fx in diameter with a fibrillar or lamellar ings of the afferent from those of the structure were seen in the terminal axo- efferent type; also, whether sensory nerves plasm (Fig. 10). Those nerves which can be distinguished from those of sym- appeared to end on connective tissue of the pathetic and parasympathetic origin when trabecular sheet may show the bare axon the functional and directional differences in direct contact with the collagen core, are not known beforehand. Data are ac- or a layer comparable to a basement mem- cumulating on the subject but many gaps brane may separate the bare nerve from exist. The ultrastructure of motor end the connective tissue. Those nerves which plates and synapses of the central and appeared to end in the endothelial cell peripheral nervous systems have been were separated from the endothelial cyto- fairly well established. Sensory receptors, plasm by an intercellular space of 150 A. however, seem to have no set pattern of In this respect they differed from nerves morphologic standards, such as those of en route through the endothelial cell only motor and associational synapses. In some in the exceptional accumulation of mito- receptors, either vesicles, or vesicles and chondria and vesicles, which suggested a mitochondria, may or may not be present synapse. It would be necessaiy to make in the pre- and postsynaptic terminal. serial sections to establish the terminal Whitearls found no striking accumula- nature of these nerves. tion of synaptic vesicles in the terminal axoplasm of corneal nerves. The only dis- Discussion tinctive feature of these afferent nerves was This study presents additional evidence the presence of rather large tubular neuro- that the majority of nerves observed in filaments in the distal portion of the nerves. the meshwork terminate here. Nerves were She suggested that these might be charac- not found to penetrate to the endothelial teristic of that part of the sensory nerve lining of Schlemm's canal although nerves fiber which is specialized for the reception were seen among the layers of endothelial of stimuli since Gray7 had found these cells immediately internal to the canal. If filaments in the visual area of the cerebral one considers the wall of Schlemm's canal cortex. Elvin,5 however, found identical to be comprised of a single layer of endo- filaments in splenic nerve endings, which, thelium, nerves were not found to pene- presumably, are efferent nerves of sym- trate the wall. pathetic and parasympathetic origin. It is not uncommon for sensoiy nerves to Neurofilaments of this size were not found be sheathed by cells other than the in trabecular nerves. Schwann cell. Sensory nerve endings have Some nerve endings of sympathetic been studied in the carotid body,15 organ of origin35 have shown vesicles which con- Corti,1(; olfactory mucosa,G Pacinian cor- tain a dense osmiophilic core which pre- puscles,12 taste buds,2 and corneal nerves.18 sumably indicates the presence of a

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reducing amine, such as norepinephrine. fibers of cat splenic nerve, J. Ultrastruct. Res. Demonstration of dense-cored vesicles in 5: 51, 1961. some of the nerve endings of the trabecular 6. Gasser, H. S.: Comparison of the structure, nerves suggests that these nerves may be as revealed with the electron microscope, and the physiology of the unmedullated fibers in peripheral neuroeffectors of the autonomic the skin nerves and in the olfactory nerves, system. Exper. Cell Res. 5: (suppl.) 3, 1958. A morphologic study such as this does 7. Gray, E. G.: Axo-somatic and axo-dendritic not reveal the functional significance of synapses of the cerebral cortex: An electron these nerves, but it provides a basis for microscope study, J. Anat. 93: 420, 1959. speculation. Since it is fairly well estab- 8. Holland, M. G., von Sallmann, L., and Col- lished that the trabecular lamellae do not lins, E. M.: A study of the innervation of the chamber angle, Am. J. Ophth. 42: 148, constitute a significant barrier to aqueous 1956. outflow, nerves ending in this area would 9. Holland, M. G., von Sallmann, L., and Col- probably be ineffective in producing a lins, E. M.: A study of the innervation of perceptive change in pressure or outflow. the chamber angle. II. The origin of trabecu- The paucity of nerves in the tissues con- lar axons revealed by degeneration experi- tiguous with Schlemm's canal would indi- ments, Am. J. Ophth. 44: 206, 1957. cate that they probably produce no great 10. Kolmer, von W.: Auge bearbeitet, in von effect at this ultimate site of aqueous out- Mollendorff, W., editor: Handbuch der mikroskopische Anatomie des Menschem, flow. Band III, Teil 2, Berlin, 1936, Julius Springer, The functional significance of these p. 243. nerves is still to be elucidated. It is possible 11. Kurus, E.: Versuch einer morphologischen that they are involved in small adjustments Analyse der Funktion und Dysfunktion der of the trabecular tissue to changing en- intraokularen Druckregulierung, Klin. vironment. Nerves which pass from one Monatsbl. Augenh. 132: 201, 1958. trabecular lamella to another through the 12. Pease, D. C, and Quilliam, T. A.: Electron microscopy of the Pacinian corpuscle, J. aqueous humor are subject to some move- Biophys. & Biochem. Cytol. 3: 331, 1957. ment, and, perhaps, stimulation as the 13. Pease, D. C, and Pallie, W.: Electron mi- ciliary muscle contracts. The physiologic croscopy of digital tactile corpuscles and relationship, if any, of the nerve endings small cutaneous nerves, J. Ultrastruct. Res. to the unique 1,000 A-banded material of 2: 352, 1959. 14. Robertson, J. D.: Preliminary observations on the lamellae is intriguing and unexplain- the ultrastructure of a frog muscle spindle, able. in Sjostrand, F. S., and Rhodin, J., editors: Proceedings of the Stockholm Conference on I wish to thank Miss Miriam Duschkin for her Electron Microscopy, New York, 1957, valuable technical assistance. Academic Press, Inc., pp. 197-200. REFERENCES 15. Ross, L. L.: Electron microscopic observations of the carotid body of the cat, J. 1. Boucheron, C. R.: Nerfs de l'hemisphere Biophys. & Biochem. Cytol. 6: 253, 1959. anterieur de l'oeil, Compt. rend. Soc. de 16. Smith, C. A., and Dempsey, E. W.: Electron biol. Par., 1889, pp. 71-78. microscopy of the organ of Corti, Am. J. 2. De Lorenzo, A. J.: Electron microscopic ob- Anat. 100: 337, 1957. servations on the taste buds of the rabbit, 17. Vrabec, Fr.: L'innervation du systeme J. Biophys. & Biochem. Cytol. 4: 143, 1958. trabeculaire de Tangle irien, Ophthalmologica 3. De Robertis, E., and Pellegrino de Iraldi, A.: 128: 359, 1954. A plurivesicular component in adrenergic 18. Whitear, M.: An electron microscope study nerve endings, Anat. Rec. 139: 299, 1961. of the cornea in mice, with special reference 4. De Robertis, E., and Pellegrino de Iraldi, A.: to the innervation, J. Anat. 94: 387, 1960. Plurivesicular secertory processes and nerve 19. Wolter, J. R.: The trabecular endothelium, endings in the pineal gland of the rat, J. A. M. A. Arch. Ophth. 61: 928, 1959. Biophys. & Biochem. Cytol. 10: 361, 1961. 20. Wolter, J. R.: Neuropathology of the trabecu- 5. Elvin, L. C: Electron-microscopic investi- lum in open-angle glaucoma, A. M. A. gation of filament structures in unmyelinated Arch. Ophth. 62: 99, 1959.

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Discussion tinguished in the electron micrographs. Similarly, Dr. Ludwig von Sallmann, Bethesda, Md. Miss preliminary histochemical examination of the Feeney's elaborate study contains important and trabecular area in our laboratory by David Paton, new information on the fine anatomy of an area, who used Koelle's technique, failed to differenti- the morphology of which is closely connected ate between cholinergic and adrenergic nerve with the problem of the physiology and pathology endings. of intraocular pressure. How new the information Miss Feeney pondered the subject of the dis- is can be illustrated by the fact that only 2 years tribution of nerves in the trabecular meshwork ago electron microscopists working on the ultra- area relative to function. In this context, the structure of the trabecular meshwork had not obvious close contact between nerve endings and observed or identified any nerves in their ma- endothelial cells might deserve more consideration terial. Now we learn from Miss Feeney's descrip- as long as these cell linings are thought by some tion and from the beautiful documentation by to influence facility of outflow. Obviously, the old good electron micrographs that nonmyelinated question referring to the function or purpose of nerves and nerve endings are frequently seen in this rich innervation cannot be settled con- most parts of the trabecular meshwork, often ad- clusively. Studies by Lele and Grimes on afferent joining endothelial cells. The outermost part of impulses induced by intraocular pressure changes the meshwork, where Schlemm's canal is located, emitted from sensory nerves in the orbit suggest and a portion of the sclera constituting the outer that the signals are not abolished when the wall of the canal were apparently devoid of nerves trabecular tissue is removed. But there is a pos- or nerve terminals. On this point, there seems to sibility that the impulses travel in the numerous be disagreement between the light microscopic small unmyelinated fibers which are not amenable observations of Holland and co-workers and the to electrophysiologic studies. Thus, the gap be- results of the electron microscopic studies. In tween morphologic detail and physiologic event preparations stained with silver and methylene cannot be bridged and there is no hope that blue some nerve terminals were seen to approach such a bridge can be built in the near future. closely the endothelial lining of Schlemm's canal. This is no way detracts from the great value I cannot offer any explanation for this discrepancy of Miss Feeney's contribution. We are all grateful of results. Many new details on the topography to her for what we learned from her excellent and morphology of the nerves of the human presentation. trabecula based on histologic studies are added Miss Feeney (closing). I am very grateful to to our knowledge of the anatomy of the region, Dr. von Sallman for his interesting discussion for instance, that the nerves frequently pass and kind comments on my paper. Regarding the freely through the trabecular space from one discrepancy in the finding of nerve endings on lamella to another, that the cytoplasm of Schwann the endothelial cells of Schlemm's canal, I can cells form the neurolemmal covering of the nerves, and that, occasionally, nerves or their endings suggest two possibilities: perhaps I did not indent or invaginate the cell membrane of endo- examine a sufficiently large number of sections thelial cells, but are, in fact, located extracellu- to find these endings, or perhaps the difficulty lies larly. The analysis of the fine structures of nerve in the differences in technique, specifically, thick- terminals is most welcome. Their appearance as ness of section. When one is viewing a 10 ft thick- free-ending axons confirms our previously held section or a whole mount, a nerve may appear to concept. Parasympathetic, sympathetic, and sen- be ending directly on the endothelial cell, but, sory nerve endings could not be clearly dis- with a 400 fold decrease in section thickness, the nerve ending may be 7 fi away.

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