The nerve-fiber layer of the primate retina: an autoradiographic study

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1 The nerve-fiber layer of the primate retina: an autoradiographic study Thomas E. Ogden The intraretinal path taken by fibers of the arcuate bundle was studied in four Rhesus monkeys. The fibers incorporated radioactivity subsequent to the injection of a small amount of S H- proline into the retina. The classical arciform projection was described and it was noted that near the disc, where the nerve-fiber layer contained many unlabeled fibers from the region of the area centralis, the label was uniformly distributed across the thickness of the layer. It is concluded that a vertical topographic organization is lacking in the nerve-fiber layer, i.e., the vertical position of a fiber within this layer, near the disc, bears no relation to the distance of its cell of origin from the disc. Key words: primate, retina, nerve-fiber layer, autoradiography, anatomy. -Lhe arcuate scotoma, so characteristic of glaucoma, represents loss of visual function within the retinal equivalent of the "Bjerrum area," that portion of the retina contributing nerve fibers to the arcuate nerve bundle. This region extends from the superior temporal or inferior temporal margin of the optic disc, sweeping temporally above or below the area centralis and widening as it extends to the far temporal periphery. From the Departments of Neurology and Physiology, University of Utah College of Medicine, Salt Lake City, Utah This research was supported by National Institutes of Health Research Grant EY and RCDA NS Manuscript submitted for publication July 16, 1973; manuscript accepted for publication Aug. 9, Reprint requests: Thomas E. Ogden, University of Utah College of Medicine Department of Neurology, Building 453, Salt Lake City, Utah Arcuate scotomata may result from a variety of diseases which interrupt one or more more fascicles of the arcuate nerve bundle, 1-2 but in such cases, and when the bundle defect is severe, they extend from blind spot to far periphery. In glaucoma, by contrast, the arcuate scotomata develop as islands, or "nuclei" within the Bjerrum area which may eventually coalesce as the disease progresses. The nuclei rarely begin at the blind spot, and when a fully developed arcuate scotoma extends to the blind spot it is always most dense in the far periphery. 3 The pathophysiology of visual loss in glaucoma is unknown, and the general belief that the primary lesion is a nerve-fiber defect at the optic nerve head, although reasonable, lacks experimental support. At least it is not clear why, if the disease process is localized to the optic disc, nerve fibers with a long, rather than a short, intraretinal path should be particularly prone to destruction. It has been suggested that ganglion cell fibers from far periphery

2 96 Ogden Investigative Ophthalmology February 1974 NFL GCL INL Fig. 1. Autoradiographs of the retina of a Rhesus monkey photographed from one tissue section at 0.5, 1, 1.5, 3.5, 6, and 7 mm. (A through F) from the optic disc. Grains overlie the full retinal thickness near the injection site (E and F). On a straight line from the injection site to the disc (C and D), there is little labeling since the arcuate fibers curve away from the area centralis. Near the disc (A, B, and C) the section crosses the arcuate bundle and the nerve-fiber layer is intensely labeled. Note the uniform distribution of grains throughout the vertical extent of the layer. NFL, nerve-fiber layer; GCL, ganglion-cell layer; INL, inner-nuclear layer. Calibration, 25fi. come to overlie those from close to the disc.4 Thus the resistance of more central retina to the effects of glaucoma could be due to the position of the more central nerve fibers, deep within the nerve-fiber layer, close to the disc. However, the vertical disposition of optic nerve fibers within the optic nerve-fiber layer of the primate is unknown. Thus it was the purpose of this study to investigate the position within the nerve-fiber layer of fibers emanating from a small peripheral region of retina contributing to the arcuate bundle in Rhesus monkeys. This was accomplished with the use of a radioisotope tracing technique described in detail by Cowan and co-workers/' The results suggest that nerve fibers of the arcuate bundle which originate in the far periphery are extensively intermingled with those that originate close to the disc. Thus differential sensitivity of different portions of the retina to the effects of glaucoma may not be ascribable to topographic organization of fibers within the nerve-fiber layer. Method Four adult Rhesus monkeys of both sexes were used in this study. The animals were tranquilized with Sernylan {0.1 mg. per kilogram) and anesthetized with 2 per cent thiopental given intravenously. Pupils were dilated with cyclopentolate hydrochloride. The outer canthus of the right eye was incised laterally to permit resection of the conjunctiva at the temporal edge of the limbus. This permitted insertion of a 17-gauge hypodermic needle into the vitreous chamber. The needle was attached to a special hydraulic electrode assembly

3 Volume 13 Number 2 Nerve-fiber layer of primate retina 97 described previously. 0 A glass capillary pipette with a beveled tip about 1M in diameter was advanced into the posterior chamber through the hypodermic needle channel, under direct visual control. The pipette was filled with 3 H-proline (10 MCi per microliter) dissolved in 1 per cent methylene blue and connected to a 5 ML syringe through fine flexible stainless-steel tubing. Contact of the pipette with the retina was easily detected ophthalmoscopically. The pipette was advanced 25M beyond the point of contact and 0.1 ML of 8 H-proline was injected. This was visible as a very small blue spot which remained confined to the region of the pipette tip. The pipette was withdrawn, the wound closed, and recovery allowed. The animals were killed five (two), six (one), and seven (one) days after the injection. The retinae were removed from the pigment epithelium and fixed flat in 10 per cent neutral formalin. The entire retina was processed for paraffin embedment and serially sectioned at 8M. The temporal retinal segment was sectioned on a plane that passed through the optic disc and fovea. Sections from every 250M across the retinae were processed for autoradiography according to the method outlined by Cowan and co-workers, 5 and exposed for 14 days. After development and fixation the sections were counterstained through the emulsion with thionin. The following abbreviations are used in this paper: NFL (nerve-fiber layer); GCL (ganglion-cell layer); IPL (innerplexiform layer); INL (inner-nuclear layer), and RL (receptor layer). Results At the time of death, the location of the injection was visible as a small dark spot about 3 mm. temporal to the fovea and 1 mm. superior to the horizontal meridian. The site of injection was easily identified on the developed slides; all retinal layers were heavily labeled. Fig. 1 shows photomicrographic montages of the retina of one of the animals. The photographs were obtained from the same tissue section at various distances from the optic disc. This section was parallel to and about 1 mm. above the horizontal meridian. Fig. 1 F shows the appearance of the injection site, 7 mm. from the optic disc; grains are evident throughout the thickness of the neural retina. Fig. 1 E, 6 mm. from the disc, shows grains most prominently in the IPL. Fig. 1 D, from area centralis 3.5 mm. from the disc, shows only a few grains in the IPL. As is characteristic of the retina in this NFL' ) OPTIC DISC } FOVE* Fig. 2. Isodensity plots of silver grain distribution in nerve-fiber layer (NFL), inner-plexiform layer (IPL), and receptor layer (RL) of the monkey illustrated in Fig. 1. Counts were made at 250M intervals in successive cross-sections. Vertical scale in millimeters also applies to horizontal dimensions. The location of the fovea and optic disc are so indicated. Contour lines, from outside in, represent 10, 20, 40, 60, 80, and 100 or more grains per 1,600M 2 - area, the NFL is very thin; the GCL and INL are relatively thick. Figs. 1 A, 1 B, and 1 C, at 0.5, 1, and 1.5 mm. from the disc, show the rapid thickening of the NFL as the disc is approached. Many grains are evident near the disc, since the section passed through the arcuate bundle. Note particularly that the grains are distributed throughout the thickness of the NFL. The photomicrographs of Fig. 1 were obtained at the position labeled 1 mm. in Fig. 2. The lateral extent of labeling was least in the RL, rather more extensive in the IPL, and followed the course of the arcuate bundle in the NFL. These relationships are illustrated in Figs. 2 and 3 which are isodensity plots reconstructed from grain counts made at 250/u. intervals over the affected areas from two of the animals. The outermost contour line represents ten grains per l,600ju, 2 above background (0 to 4 grains per 1,600/x 2 ). The subsequent lines represent 20, 40, 60, 80, and 100 or more grains per 1,600/x 2. At each position the grains overlying the particular layer (RL,

4 98 Ogden Investigative Ophthalmology February 1974 Fig. 3. Isodensity plots of silver grain distribution in NFL, IPL, and RL of second monkey. Intraretinal spread of the label was greater than that of the first monkey, but the course of the arcuate bundle is clearly evident. See Fig. 2 legend for details. IPL, or NFL) were counted, normalized for variations in thickness of the layer, and tabulated as grains per 1,600/A 2. All counts were made at 430x magnification. Fig. 2 represents the most localized injection. The results of the experiment shown in Fig. 3, and from the two other animals, support the findings shown in Fig. 2, but less neatly since the spread of the label was somewhat more extensive. In each case the center of the injection site was oval, and measured about 250 by 500JU,. This was most sharply represented in the receptor layer where grain density declined rapidly with distance from the center. Diffusion of label in the IPL was uniform except in the direction of the area centralis which was unlabeled in all animals. The contours of the arcuate bundle are clearly evident. Thus this study elegantly reveals the origin and course of the fibers of the arcuate bundle and confirms the traditional view of its relationships. 4 Consider the NFL isodensity plot of Fig. 2. The entire upper half of the area centralis contributes unlabeled fibers to the

5 Volume 13 Number 2 Nerve-fiber layer of primate retina 99 NFL; these greatly increase the thickness of this layer as the disc is approached (Figs. 1 A, 1 B, and 1 C). If these added fibers simply displaced the more peripheral (labeled) fibers to the surface, the label of the NFL would be confined to a thin superficial lamina. That this is not the case is obvious (Fig. 1 A). Apparently there is extensive intermingling of central with peripheral nerve fibers throughout the thickness of the NFL. It should be noted that the NFL at the disc margin is nearly 75/A thick (Fig. 1 A). The beta particles emitted by the incorporated tritiated amino acid in the labeled fibers has a tissue penetrance of only a few micra, therefore the even distribution of grains across the NFL cannot be the result of lack of resolution of the autoradiographic method (see also 7). Clearly, within the nerve-fiber layer adjacent to the optic disc, labeled nerve fibers of the arcuate bundle from the far periphery are intermingled with unlabeled fibers whose cells of origin are close to the area centralis. It was hoped, when this study was undertaken, that evidence for the existence of "association" ganglion cells might be found. Gallego and Cruz 8 observed such cells in Gros silver preparations of the retina of the dog. These were ganglion cells, particularly in peripheral retina, whose axons terminated in the IPL at some distance from their somata. The presence of such cells in the monkey might be revealed in the present study as a concentration of grains over the IPL, some distance from the injection site. In the experiment illustrated in Fig. 3, there was widespread labeling of the IPL which decremented gradually in all directions, presumably as a result of diffusion of the 3 H-proline. Five small regions were observed where the grain density was twice that of the neighboring regions. These regions might represent concentrations of label in the axon terminals of association ganglion cells, but this is unlikely and the evidence is inconclusive since it was observed in only one of the animals. Additional experiments with more localized injections will be required to establish the presence of ganglion cells with intraretinal projections in the primate. Discussion This study has shown the facility with which the autoradiographic method can be used to demonstrate a fiber pathway, and has revealed a lack of vertical topographic organization within the nerve-fiber layer of the primate retina. Yet the classic study of the arcuate bundle of Rhesus monkeys by Hoyt" concluded "retinal fibers arising from peripheral retina pass into the optic nerve nearer its surface. Fibers arising from those ganglion cells close to the disc pass-up the nerve nearer its core." He studied degenerating fibers with the Nauta technique and did not describe the distribution of fibers within fascicles at the edge of the disc. However, the results of his study imply a topographic organization with fibers from the far periphery lying beneath those arising close to the disc. This possibility was appreciated by Polyak 4 who states "the extreme peripheral fibers, originally superficial, may gradually sink deeper and be the first to slip into the papilla, in this way leaving the fibers from regions closer to the central fovea and area on the papillar surface and thus bring them closer to the center of the papilla." From the present study it is apparent that nerve fibers intermingle freely along the intraretinal course of the arcuate bundle; segregation of fibers to bring those from far periphery to the outside and those from nearer the disc to the center of the optic nerve must occur as, or immediately after, the fibers cross the disc edge. There seems to be general agreement in the literature that the loss of retinal function associated with glaucoma results from inadequacy of perfusion of the optic-nerve head. 1 ' 10 The peculiar susceptibility of the most peripheral fibers of the arcuate bundle to the effects of glaucoma might result from their position within the optic nerve below the lamina cribrosa, but it seems unlikely that their organization at the disc

6 100 Ogden Investigative Ophthalmology February 1974 margin which is apparently random, could be involved. Intraretinal nerve fibers are unmyelinated in the primate and often lie adjacent to one another without intervening glia. It is possible that the intermingling of fibers as they course toward the disc acts to prevent ephaptic excitation of adjacent fibers, a phenomenon which would be destructive of visual efficiency. I thank W. F. Hoyt and H. J. L. Van Dyk for helpful discussions of this project and I gratefully acknowledge the assistance and advice rendered me by W. M. Cowan and others of the Department of Anatomy, Washington University, St. Louis, Mo. REFERENCES 1. Harrington, D. O.: The pathogenesis of the glaucoma field, Am. J. Ophthalmol. 47: 177, Harrington, D. O.: The Visual Fields, St. Louis, 1971, The C. V. Mosby Company. 3. Drance, S. M.: The glaucomatous visual field, Br. J. Ophthalmol. 56: 186, Polyak, S.: The Vertebrate Visual System, Chicago, 1957, University of Chicago Press. 5. Cowan, W. M., Gottlieb, D. I., Hendrickson, A. E., et al.: The autoradiographic demonstration of axonal connections in the central nervous system, Brain Res. 37: 21, Ogden, T. E., and Brown, K. T.: Intraretinal responses of the cynamolgus monkey to electrical stimulation of the optic nerve and retina, J. Neurophysiol. 27: 682, Rogers, A. W.: Techniques in Autoradiography, New York, 1967, Elsevier. 8. Gallego, A., and Cruz, J.: Mammalian retina: associational nerve cells in ganglion-cell layer, Science 150: 1313, Hoyt, W. F.: Anatomic considerations of arcuate scotomas associated with lesions of the optic nerve and chiasm. A Nauta ax on degeneration study in the monkey, Bull. Johns Hopkins Hosp. Ill: 57, Hayreh, S. S.: Blood supply of the optic nerve head and its role in optic atrophy, glaucoma, and oedema of the optic disc, Br. J. Ophthalmol. 53: 721, 1969.