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Reports 1045 Volume 13 Number 12 vestigation applying this method to the experimentally damaged retina (monkey) is in progress in our laboratory. In the present study, the Miiller cells, seen in the horizontal section of the retina, were often found in a small mass in close contact with the neighboring cells. This tendency is more noticeable in the posterior than in the peripheral part of the retina. Larger masses of the Miiller cell processes have been observed in the extremely thickened nerve fiber layer in the juxta-optic nerve region of the human retina.s These facts may represent functional aspects of the Miiller cells such as mechanical support of the retina. The present observations also showed quite clearly that there is a close relationship between the retinal capillary and the Miiller and accessory glial cells and are in accordance with those of earlier investigators. The astrocytes, terminating on the surface of the capillary, always showed an end-foot structure comprising bundled processes. The light microscopic studies"- 1() of the retinal glia have revealed the presence of an additional two types of glial cells, perivascular glia and lemmocyte (cell of Remak), but these glial elements have not been pursued in this study. We would like to extend our sincere thanks to Victoria Ozanics for her valuable advice and reviewing this manuscript. From the Department of Ophthalmology, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Submitted for publication April 26, 1974. Reprint requests: Dr. S. Uga, Department of Ophthalmology, Faculty of Medicine, Kyushu University, Fukuoka, japan. REFERENCES 1. Stell, W. K.: Correlation of retinal cytoarchitecture and infrastructure in Golgi preparations, Anat. Rec. 153: 389, 1965. 2. Dowling, J. E.: Organization of vertebrate retinas, INVEST. OPHTHALMOL. 9: 655, 1970. 3. Matsusaka, T.: The fine structure of the inner limiting membrane of the rat retina as revealed by ruthenium red staining, J. Ultrastruct. Res. 36: 312, 1971. 4. Peyman, G. A., Spitznas, M., and Straatsma, B. R.: Peroxidase diffusion in the normal and photocoagulated retina, INVEST. OPHTHALMOL. 10: 181, 1971. 5. Ashton, N., and Tripathi, R.: The argyrophilic mosaic of the internal limiting membrane of the retina, Exp. Eye Res. 14: 49, 1972. 6. Magalhaes, M. M., and Coimbra, A.: The rabbit retina Miiller cell. A fine structural and cytochemical study, J. Ultrastruct. Res. 39: 310, 1972. 7. Luft, J. H.: Fine structure of capillary and endocapillary layer as revealed by ruthenium red, Fed. Proc. 25: 1773, 1966. 8. Uga, S.: Some structural features of the retinal Miillerian cells in the juxta-optic nerve region, Exp. Eye Res. 19: 105, 1974. 9. Wolter, J. R.: Perivascular glia of the blood vessels of the human retina, Am. J. Ophthalmol. 44: 766, 1957. 10. Lessel, S., and Kuwabara, T.: Retinal neuroglia, Arch. Ophthalmol. 70: 133, 1963. Estimation of the ratio of cones to neurons in the fovea of the human retina. L. MisSOTTEN The ratio of pedicles over neurons has been determined in thin sagittal and -flat sections of the rod-free central area of the human retina. The real amount of neurons has been calculated from the number of transections of nuclei. Results show that for each pedicle two to three bipolar cells are found, ± 0.6 horizontal cells, t 0.7 amacrine cells, and 0.9 ganglion cells. The central rod-free area of the fovea is especially well-suited for a study of the synaptic contacts of the cones. Only one class of receptor cells is present in this area and the neurons of the second and third echelon are small and, for this reason, easier to investigate by electron microscopy. In order to translate the findings of the analysis of single cells in a general schema, it would be helpful to know the ratio between the different neurons and the receptor cells. A comparison of the data available from the literature on the number of cones1 and the number of ganglion cells- does not give the answer, because the inner layers of the retina are shifted centrifugally in respect to the visual cells (Fig. 1). In front of the central bouquet of cones, in the foveola, the inner layers of the retina are absent. No synaptic connections are found in this area. The synaptic pedicles of these cones are located in an annular zone surrounding the central area; they are connected to their parent cells with long fibers "Henle's fibers." The centermost pedicle is usually found at 100 microns distant from the center of the fovea. The area between 100 and 300 microns contains pedicles at irregular intervals; from about 300 microns outward the pedicles form a continuous and uniform layer to about 800 to 1,000 microns from the center, where rod spherules are found in increasing numbers. The centrifugal shift of the neurons connected to the cones of the fovea hinders the correlation of the numbers of receptor cells and neurons, because the layers are displaced in respect to each other, and because the area available to the neurons is much larger than the surface occupied by the cones, as is shown in Fig. 1. The neurons of the inner layers are oriented Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017 1046 Investigative Ophthalmology Deceviber 1974 Reports Qfi 500M. Fig, 1. The centrifugal shift of the pedicles of the cones of the fovea as seen in flat section (upper drawing) or in sagittal section (below). A, the 2,500 cones of the central bouquet (dark area) have their pedicles situated in an annular area (grey) extending from 125 microns to 260 microns from the center. B, the 25,000 cones of the fovea externa (dark) and their pedicles (grey area). C, the 100,000 cones of the slope of the fovea (dark area) and their pedicles (grey area). This schematic representation shows how the area covered by the pedicles is several times larger than the area of the receptors. The lower schematic drawing shows how the neurons of the second and third echelon are placed in front of the pedicles. The scale applies to retina fixed in glutaraldehyde dichromate and embedded in epon. perpendicular to the surface of the retina, as can be seen very clearly in a Golgi-stained preparation. The proportion of neurons to cones may thus be determined by counting pedicles and neurons in the area between 300 and 800 microns from the center of the fovea. This approach has been used by Vilter,a but this author does not mention the horizontal cells nor the amacrine cells in his paper and does not take into account the stereologic problems. Methods. Human retinae have been obtained from eyes with normal visual function enucleated for melanomas of the iris root or the ciliary body. They have been prepared for Golgi staining by the glutaraldehyde-dichromate method,4 dehydrated, and embedded in Epon, and cut in 60micron thick sections on a sliding microtome after softening the superficial layers of the block with heat.5 The fovea was located, and Golgi-stained cells recorded in optical micrographs. The central area was then remounted and cut on an ultra microtome in thin sections for electron microscopy and in one micron-thick sections for optical microscopy. Sagittal sections through the center of the fovea have been made, together with "flat" sections parallel to the surface of the retina from an area situated at 600 to 800 microns from the center. Measurements and counting was done on micrographs made with a Zeiss Photomicroscope with a 40x oil immersion planapochromate and studied at a final magnification of l,000x (Fig. 2). Pedicles and neurons have been counted in tangential and flat sections. The number of neurons has been estimated by counting their nuclei. All dimensions apply to embedded retina. No correction was made for the shrinkage during fixation and embedding. The real number of nuclei in a unit volume (Ni) has been calculated from the apparent number of nuclei (Na) in a unit area Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017 Volume 13 Number 12 Reports -wt« Fig. 2. A sagittal section of human retina, situated at 550 microns from the center of the fovea. From top to bottom: the outer plexiform layer with Henle's fibers and pedicles, the inner nuclear layer, the inner plexiform layer, the layer of ganglion cells, the layer of optic nerve fibers, and the inner limiting membrane. The mark measures 50 microns. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017 1047 Investigative Ophthalmology December 1974 1048 Reports Table I. Number of transections of pedicles and nuclei in sagittal sections Pedicles Nuclei of the inner Horizontal cells Bipolar cells Amacrine cells Ganglion cells nuclear layer 172 (15.5%) 731 (66%) 207 (18.5%) 298 1.110 495 Table II. Number of neurons in a volume of foveal retina containing 100 pedicles Neurons in the inner nuclear layer Horizontal cells 45.5- 71 Bipolar cells 194 -303 Amacrine cells 54.5- 85 Ganglion cells 294-458 90 of the thin sections by means of the relation0: Ni = Na 3/2 K (Vi/Vt) 1/2 Vi/Vt is the ratio of the volume occupied by the nuclei of the neurons to the total volume of the inner nuclear layer or, for the ganglion cells; the ratio of the volume of their nuclei to the volume of the ganglion cell layer. These ratios have been determinated by measuring their surface in the sections by means of the "point" method proposed by Glagolev.7 P equals 1.38 for spherical bodies0 and is not appreciably different for ellipsoids up to a ratio of width over a length of 0.6.s K = 1 for a population of bodies of identical size. Results. Apparent numbers of pedicles and neurons. One micron-thick sagittal sections through the center of the fovea have been studied and the number of pedicles and neurons has been determined in 19 areas, each 100 microns long, situated between 300 and 800 microns from the center. No significant differences have been found between these areas. The results have been pooled as shown in Table I. Pedicles and neurons have also been numbered on 47 flat sections, chosen at different levels in a region situated at 600 to 800 microns from the center of the fovea. Differentiation. The nuclei of different types of neurons show different forms and staining patterns (Fig. 2). Horizontal cells have dark nuclei with a few black spots, amacrine cell nuclei are grayish and have indentations, Miiller cells have very dark angular nuclei. The characteristics are not much influenced by the fixative used, be it osmium tetroxide, glutaraldehyde-osmium tetroxide, or glutaraldehyde dichromate. The bipolar cells show different aspects accord- ing to the fixative used. In osmium-fixed tissues all the bipolar cells have grayish nuclei; with glutaraldehyde-dichromate fixation two kinds of nuclei are seen in about equal numbers in bipolar cells: some nuclei are uniform grayish and others are pale with dark spots (Fig. 2). This differentiation is not equally well visible in all retinae; its significance is still unclear. The identification of the neurons by their nuclei is based on: (1) the preferential location of each type of nucleus, (2) the study of nuclei of Golgistained neurons, and (3) the correlation with electron micrographs. In some instances, the aspect of a nucleus was ambiguous: especially the amacrine cell nucleus whose indentation is not visible in the section is hard to differentiate from a gray bipolar cell nucleus. Pedicles. In sagittal sections 15.5 pedicles are counted per 0.1 mm., corresponding to 15.52 or 240 pedicles per unit volume. This number could be verified on flat sections, where 250 pedicles are found per 0.01 mm.2. The latter determination is more direct; it will be used as a base for further calculations. Inner nuclear layer. Weibel and Gomez'0 equation has been used to calculate the real number of neurons in the inner nuclear layer. In the foveal area this layer is approximately 50 microns thick. In sagittal sections the relative surface of nuclei equals 39.5 per cent and 29 transections are seen per (50 microns),2 as may be deduced from Table I. In flat sections, 41 transections are counted per unit area; their relative surface measures 43 per cent. As an approximation, we assume that the nuclei are spherical and all of the same size. The introduction of these values in the equation shows that a unit volume of (50 microns):t contains between 184 nuclei (calculated from measurements on sagittal sections) to 286 nuclei (based on flat sections). The 50 times 50 micron outer surface of this unit volume is adjacent to 62.5 pedicles. The number of neurons per 100 pedicles, and the number of different kinds of neurons is shown in Table II. The lowest number results from measurements on sagittal sections, the highest number from flat sections. Ganglion cell layer. The ganglion cell layer in sagittal sections has a mean thickness of 70 microns and 18.2 transections of nuclei are found in (70 microns).- They occupy a relative surface of 25.8 per cent. The analysis of flat sections gave identical results. Calculation of the Equation 1 shows that a unit volume of (70 microns ):t contains 110 ganglion cell nuclei or ganglion cells. The outer surface of this unit volume corresponds to an area containing 122 pedicles. Transformation of this result for a volume of retina containing 100 pedicles is shown in Table II. Discussion. Stereologic problems. Pedicles are Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017 Volume 13 Number 12 best visualized and numbered in one micron-thick sections. They form a single layer and may be counted in sagittal and flat sections. The estimation of the number of neurons from thin sections, however, requires the transformation from apparent to real numbers by means of a principle developed by Weibel and Gomez.(i These authors enumerate four conditions that must be satisfied. The investigated bodies must be randomly distributed, they must be well defined, the bodies must be small compared to the total volume, and the section must be thin. The first condition means that any section in whatever direction should pass through the same number of bodies. Although the inspection of sections such as Fig. 2 suggests that the distribution of the nuclei in the nuclear layers is more or less random, comparison to flat sections shows that in the inner nuclear layer this condition is not satisfied, whereas in the ganglion cell layer the distribution is random. For this reason, the calculated number of ganglion cells is fairly accurate. In the inner nuclear layer, calculations based on flat sections, showing the highest concentration of transections, and on sagittal sections that have the lowest density, set an upper and lower limit to the number of neurons. The coefficient fi in the equation reflects the configuration of the bodies to be counted. Coefficient /3 equals 1.382 for spheres, and grows with increasing lack of sphericity. It has been shown that, for ellipsoids with a ratio of diameter over length of 0.6 or more, the increase of fi is small,8 less than was originally calculated. As the nuclei of the retinal neurons are only slightly elongated, the value 1.382 for /S has been used. This results in a slight overestimation of the number of neurons. The coefficient K equals one for bodies of equal size, and increases with increasing heterogeneity of the sample. It has been shown that in biological material the coefficient K is usually between 1.02 and 1.1.!) The nuclei in the inner nuclear layer and those in the ganglion cell layer each form a fairly homogenous population, as can be seen in thick sections. For this reason, the coefficient K has been assumed to be one. This approximation results in a slight underestimation of the number of neurons. The incertitude introduced by these two approximations, however, is much less than that due to the lack of randomness. The synoptic connections of the cones. The discovery of the flat midget bipolar10 in addition to the invaginating midget bipolar has given rise to the hypothesis that each cone might be connected to one neuron of each type. In addition, contacts are made with the diffuse cone bipolars also present in the fovea. Reports 1049 The ratio of bipolar cells over pedicles being approximatively 2.5 confirms that this is a possibility. Direct confirmation may be obtained from electron microscopic study of Golgi-stained retina, combined with reconstruction of the unstained neurons from serial sections. However, the present amount of information accumulated by this method is still insufficient to permit generalizations. The author thanks Mrs. A. Geysen for her expert technical assistance and Miss Chr. Van Rijmenant for typing and proofreading the manuscript. From the Department of Ophthalmology, University of Louvain, K.U.L., Belgium. This investigation was supported by Grant No. 1201 from the Belgian Fund for Medical Scientific Research. Submitted for publication May 7, 1974. Reprint requests: Prof. L. Missotten, Ophthalmology, Academisch Ziekenhuis St. Rafael, Capucienenvoer, B 3000 Leuven, Belgium. Key words: retina, fovea, cones, horizontal cells, bipolar cells, amacrine cells, ganglion cells. REFERENCES 1. Oesterberg, G. A.: Topography of the layer of rods and cones in the human retina, Acta Ophthalmol. 6: 1, 1935. 2. Oppel, O.: Untersuchungen iiber die Verteilung und Zahl der retinalen Ganglienzellen beim Menschen, Albrecht v. Graefes Arch. Klin. Exp. Ophthalmol. 172: 1, 1967. 3. Vilter, V.: Recherches biom6triques sur l'organisation synaptique de la retine humaine, C.R. Soc. Biol. 143: 830, 1949. 4. Colonnier, M.: The tangential organization of the visual cortex, J. Anat. 98: 327, 1964. 5. West, R. W.: Superficial warming of epoxy blocks for cutting of 25 to 150 micron sections to be resectioned in the 40 to 90 nm. range, Stain Technol. 47: 201, 1972. 6. Weibel, E. R., and Gomez, D. M.: A principle for counting tissue structures on random sections, J. Appl. Physiol. 17: 343, 1962. 7. Glagolev, A. A.: Quantitative analysis, with the microscope by the "point" method, Eng. Mining J. 15: 399, 1934. 8. Baudhuin, P.: L'analyse morphologique quantitative de fractions subcellulaires, These Universite de Louvain, 1968. 9. Weibel, E. R., Kistler, G. S., and Scherle, W. F.: Practical stereological methods for morphometric cytology, J. Cell Biol. 30: 23, 1966. 10. Kolb, H., Boycott, B. B., and Dowling, J. E.: Organization of the primate retina, light microscopy. Appendix: A second type of midget bipolar cell in the primate retina, Phil. Trans. B. 255: 109, 1969. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017