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HILD, Department of Anatomy, Medical Branch, The University of Texas, Galveston, Texas 77550/USA Prof. Dr. J. van LIMBORGH, Universiteit van Amsterdam, Anatomisch-Embryologisch Labora torium, Mauritskade 61, Amsterdam-O/Holland Prof. Dr. R. ORTMANN, Anatomisches Institut der Universitat, Lindenburg, D-5000 Koln-Linden thai Prof. Dr. T. H. SCHIEBLER, Anatomisches Institut der U niversitat, KoellikerstraBe 6, D-8700 Wiirz burg Prof. Dr. G. TOND URY, Direktion der Anatomie, GloriastraBe 19, CH- 8006 Ziirich/Schweiz Prof. Dr. E. WOLFF, Lab. d'Embryologie Experimentale, College de France, 11 Place Marcelin Berthelot, F-75005 Paris/Frankreich Advances in Anatomy, Embryology and Cell Biology Ergebnisse der Anatomie und Entwicklungsgeschichte Revues d'anatomie et de morphologie experimentale Vol. 54 . Fasc.4 Editors: A. Brodal, Oslo· W. Rild, Galveston· 1. van Limborgh, Amsterdam· R. Ortmann, Kaln . T.R. Schiebler, Wurzburg . G. Tandury, Zurich· E. Wolff, Paris Margarete Vogel Postnatal Development of the eat's Retina With 27 Figures Springer-Verlag Berlin Heidelberg New York 1978 Dr. Margarete Vogel, Klinikum der Medizinischen Hochschule Lubeck, Abteilung fur Anatomie, Ratzeburger Allee 160, D-2400 Lubeck, Federal Republic of Germany ISBN-13: 978-3-540-08799-1 e-ISBN-13: 978-3-642-66974-3 DOl: 10.1007./978-3-642-66974-3 This work is subject to copyright. 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Stiirtz AG, Universitatsdruckerei, Wiirzburg 2121/3321-543210 Contents Introduction .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7 Material and Methods ........................................ 8 Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 1. Qualitative Morphological Results .. . . . . . . . . . . . . . . . . . . . . . . . . .. 10 1.1. Internal Limiting Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 1.2. Nerve Fibre and Ganglion Cell Layer ...................... 10 1.3. Inner Plexiform Layer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13 104. Inner Nuclear Layer ................................. 16 1.5. Outer Plexiform Layer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 18 1.6. Outer Nuclear Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 22 1.7. External Limiting Membrane ........................... 23 1.8. Inner and Outer Segments ............................. 23 1.9. Pigment Epithelium ................................. 30 1.10. Choriocapillaris and Bruch's Membrane ..................... 31 1.11. Tapetum Lucidum Cellulosum .......................... 32 2. Quantitative Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 36 2.1. General Remarks ................................... 36 2.1.1. Morphometric Results . . . . . . . . . . . . . . . . . . . . . . . . . .. 36 2.1.2. Determination of Layer Thicknesses by Light Microscope ... 37 2.1.3. Comparison of Results Obtained by Light-and Electron Microscope ...... . . . . . . . . . . . . . . . . . . . . . . . . . . .. 39 2.2. Discussion of the Quantitative Results ..................... 39 2.2.1. Non-Nerve Retinal Structures . . . . . . . . . . . . . . . . . . . . .. 39 2.2.1.1. Retinal Glia . . . . . . . . . . . . . . . . . . . . . . . . . . .. 40 2.2.1.2. Retinal Blood Vessels ..................... 40 2.2.2. Intercellular Spaces. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 41 2.2.3. Retinal Structures Outside the External Limiting Membrane.. 41 2.204. Nuclear Layers of the Neural Retina. . . . . . . . . . . . . . . . .. 42 2.204.1. Outer Nuclear Layer . . . . . . . . . . . . . . . . . . . . .. 42 2.204.2. Inner Nuclear Layer ...................... 43 2.204.3. Ganglion Cell Layer ...................... 44 2.20404. Comparison of the Retinal Nuclear Layers. . . . . . .. 45 2.2.5. Neuropile Layers of the Retina . . . . . . . . . . . . . . . . . . . .. 46 2.2.5.1. Outer Plexiform Layer. . . . . . . . . . . . . . . . . . . .. 46 2.2.5.2. Inner Plexiform Layer ..................... 47 2.2.5.3. Nerve Fibre Layer ........................ 47 2.2.5 A. Comparison of the Neuropile Layers ........... 47 2.2.6. Total Thicknesses of Retinae ...................... 48 5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 48 Summary ................................................ 57 References ............................................... 59 Subject Index ............................................. 65 6 Introduction The retina as an organ of perception of light, colour, shape and movement has been the subject of numerous and intensive light-and electron-microscopical investigations. To date the interest in these has largely been concentrated on the structure of the ma ture retina and the genesis of its cellular elements. The first exhaustive observations on the development of the retina in vertebrates were made by Babuchin (1863). Using the retinae of chicken embryos, he showed that Millier's radial fibres and the ganglion cells are the first to develop, while the receptor segments are the last. Subsequently, the early differentiation of Millier's radial fibres was often reaffirmed (Cajal, 1893; Meller, 1968; Bhattacharjee and Sanyal, 1975; and others). Furthermore, Babuchin had already indicated that the structural development in the area of the posterior pole is very rapid compared with those regions of the retina which are situated more peripherally. Today, when comparing results of electron-mi croscopical investigations, this fact is of particular importance, since in each case only very limited areas of the retina can be examined. Schultze (1867a, b) pointed out the uniformity of origin and the general classifica tion of light-perceiving elements into inner and outer segments, thus contesting the hitherto generally held opinion that these structures, like the pigment epithelium, ori ginate from the outer leaf of the eye cup. In 1881 Ogneff discovered the analogous mode of formation in birds and mammals. The light-microscopical classification of retinal neurons is based to date on a system by Ramon y Cayal (1893,1894). In his treatise "La retine des vertebres" Cajal charac terized the retina as "an organ whose structure shows remarkable uniformity amongst all vertebrates ... Only a few modifications appear in its construction, depending on the faculty of sight of each particular animal". The discovery by Cajal of the funda mentally similar construction of the cells and layers of the retina of the vertebrates has been frequently confirmed by later comparative anatomical investigations, and is to day generally recognized. With the advent of the electron microscope, however, it be came possible to show that the ultrastructure of all layers contains numerous, and of ten very subtle differences which were hitherto unknown. The histogenesis, too develops in principle according to the same basic rules (Rohen, 1964; Starck, 1975). Nevertheless many problems of detail in the morphogenesis are as yet unsolved. Thus, there is no agreement on, for example, the question of which morphological processes lead to the formation of the photolamellae in the receptor outer segments (Tokuyasu and Yamada, 1959; De Robertis, 1960; Weidman and Ku wabara, 1968; Hebel, 1971; and others), which causal and functional connections exist between the development at varying times of the neuropile layers (Weidman, 1975) or what role can be ascribed to cellular decay in the developing nuclear layers (Gli.icks mann, 1940; Kuwabara and Weidman, 1974; Rager, G. and Rager, U., 1976). Cell death was observed in the normal embryogenesis of many peripheral organs (review by Saunders, 1966) and in various regions of the central nervous system (Mattanza, 1973; Zilles et al., 1975a, b; Pilar and Landmesser, 1976; Aguayo et al., 1976). The meaning of cellular decay as a normal event in developing tissues is largely unexplained. Futher- 7 more, the scanty knowledge about the interrelations between morphological, physiolo gical and biochemical questions relating to sight should also be pointed out. This investigation, largely by electron microscope, is concerned specifically with the postnatal development of the retina of the cat. The cat is born altricially, blind, with closed eyelids, and only opens its eyes between the 7th and 10th postnatal days (War kentin and Smith, 1937; Hof - Van Duin, 1976). After birth there are phases of considerable structural differentiation. Donovan (1966) has described its postnatal de velopment using the light microscope. However, relatively few detailed electron-micros copical investigations have been carried out on the retinal differentiating processes of this species, e. g. on the outer segment genesis (Tokuyasu and Yamada, 1959 and 1960; Cragg, 1975) and on the development of the tapetum lucidum cellulosum (Bernstein and Pease, 1959, Pedler, 1963; Wolff, H. H. 1968; Btissow, 1974). Coherent morphological studies of the fundamental ultrastructural processes have so far been published only for the development of the retina of the dog (Hebel, 1971), the guinea pig (Spira, 1975), the rat (Weidman and Kuwabara, 1968; Kuwabara and Weidman, 1974; Weidman, 1975), the mouse (Olney, 1968) and the chicken (Meller, 1968). Quantitative structural data about the developmental processes of the retina exist to our knowledge neither for the cat nor for any other species. Electron-microscopical information about the mature retina of the cat, however, is abundant (Kidd, 1962; Brown and Major, 1966; Boycott and Kolb, 1973; Steinberg and Wood, 1974 and 1975; Nelson et al., 1975; Famiglietti and Kolb, 1975; and others) and various structures have been subjected to a quantitative analysis (Stone, 1965; Stone and Hollander, 1971 ; Dubin, 1971; Hollander and Stone, 1972; Steinberg et al., 1973). In the following "pilot study, " the differentiations taking place in the retina of the cat during the postnatal period will be considered and will be presented, on an elec tron-microscopical basis, both in a qualitative morphological and in a quantitative mor phometric context. Material and Methods The development of the postnatal maturation of the retina of the cat in the region of the posterior eye pole was examined throughout 17 stages of development from neonatal to adult (Table 1) (col lection of material by Haug, 1972). For seven of the retinae (6 h; 5, 8,15, 30, 62 days and adult animal) morphometric measurements of the relative volumes of the different cellular structures were carried out. Preparation of material to be examined. The cat's eyes, fixed by perfusion (5 % glutaraldehyde buffered to pH 7.4; Haug, 1971), were removed, halved by making a mediosagittal cut, and cres cent-shaped tissue sections, about 1 - 1.5 mm wide, bordering temporally on the optic papilla, were cut out. After 12 h in a phosphate buffer mixed with saccharose (0.22 m), the strips of retinal tissue were fixed in a 4 % OsO. solution and embedded in Araldite. Semi-thin and ultra-thin sec tions were produced from the retinal area directly bordering on the temporal site of the optic nerve (length of edge about 1 mm), using glass knives on the Reichert Ultra-microtome OM U3. The sec tions, cut in the temporal-nasal direction, were made at right angles to the inner surface of the re tina. Semi-thin sections were stained with azure II-methylene blue solution (Richardson et aI., 1960). Ultra-thin sections were supported on slotted grids coated with zapon enamel, with the in ner limiting membranes of the retinae oriented at right angles to the bars. After double staining with uranyl acetate and lead citrate (Echlin, 1964), the sections were examined with the Zeiss EM 8 Table 1. List of the eat retinae examined (BW, body weight; BrW, weight of brain; qual. eval., morphological evaluation and measurement of layer thickness; quant. eval., morphometric evaluation from electron micrographs) Age Serial Sex BW BrW Qual. Quant. number (grams) (grams) eva!. eval. 6h 914 d 89 4.1 + + 12 h 928 d 81 4.5 + 2 days 912 <;I 154 6.8 + 3 days 925 d 185 6.5 + 5 days 938 <;I 208 7.9 + + 7 days 911 <;I 203 8.5 + 8 days 926 d 270 8.9 + + 12 days 939 <;I 258 10.1 + 14 days 933 d 264 12.6 + 15 days 916 <;I 302 14.0 + + 17 days 934 <;I 302 12.4 + 22 days 927 d 486 16.8 + 30 days 918 d 419 19.5 + + 42 days 920 <;I 610 23.5 + 62 days 923 <;I 1010 26.8 + + 136 days 943 d 2300 31.7 + adult 946 <;I 3200 27.2 + + 9 S-2 electron microscope. For the quantitative studies, five coherent series of photographs per animal were made, of which the positives, enlarged 7500 times, were stuck together on montages (width approx. 40 em, length 140 -200 cm). Determination of volume parts and their calculation. The volume parts of the retinal structures (X, Y, Z, say) were determined by means of the point-counting technique according to the for mula: P (X) + P (Y) + P (Z) = P (total) = V (total) = 100 % (Chalkley, 1943; Haug, 1955 and 1962; Hennig, 1957). For the evaluations, a rectangular measuring grid (36 x 6 cm) with 4 horizontal and 25 vertical lines, i. e. 100 uniformly spaced measuring points, was used. The rectangular shape of the evaluation grid, which was oriented with its longitudinal axis parallel to the inner surface of the retina, was intended to ensure a representative collection of data from relatively narrow layers. Thus on the one hand, the low height of the grid increased the selectivity of the layers, whilst on the other, the random variations in density of the structures, which could be observed longitudinal ly within the measuring fields, were evened out. The number of measuring fields for each age examined was the same. The counts were recorded using the Kontron M.O.P. KM II. Each of the five composite photographs for a particular stage of development was evaluated five times, so that for each animal 25 series of measurements, noted as rows, were obtained, each consisting of 18 -22 lines of values corresponding to the respective number of measuring fields arranged parallel to the inner surface of the retina. Twelve different structural details could be evaluated corresponding to the 12 registers of the M.O.P. KM II. How ever, some of the registers could be doubly allocated where the associated cellular elements in the retinal layers investigated did not overlap each other. Altogether between 45,000 and 55,000 points per animal were available for the calculations. The arrangement of the measuring fields in lines and the counted structures in rows facilitated a relatively simple calculation of results for each layer and totals for all layers (Haug et aI., 1971). By means of a four-part computer program, written in BASIC, the calculation of mean values for all structures could be carried out with their confidential limits (0< = 0.05 or 0.01). Several of the structures for which values were initially recorded separately could be combined later on by totall ing (e. g. inner and outer receptor segments or the sum of all nerve elements, etc.). When collecting data, the counting points lying ou tside the retina were recorded in a separate register. These un- 9 wanted values could be eliminated by means of a special program step. Thus the values of 100 vol % refers exclusively to the structures lying within the retina. Measurements of thickness of retinal layers. These were made on semi-thin sections for each of the 17 animals, using a Zeiss screw micrometer eyepiece. Results 1. Qualitative Morphological Results The various cell elements, which sometimes show considerable morphological differ ences at the various ages, will be dealt with layer by layer, proceeding from the inner limiting membrane towards the tapetum. Figure 1 shows an overall view of the retinal structures and layers of the mature retina and the neighbouring choroid with the choriocapillaris and the tapetum lucidum, as a demonstration of its final state of de velopment. On account of its functional significance for the process of vision, the differentia tion of the tapetum lucidum cellulosum, which lies outside Bruch's membrane, is also taken into consideration in the discussion of qualitative results. 1.1. Internal Limiting Membrane Forming the boundary of the retina with the vitreous body, and resembling a lamina basalis (Wolff, E., 1968), the internal limiting membrane is formed at an early embry onic stage during the development of the eye cup (Kuwabara and Weidman, 1974)_ Es sentially it is a product of the retinal glia, i. e. the Miiller cells, and, to a lesser extent, of the astrocytes. Vitreous fibrils and mucopolysaccharides play only an insignificant role in its formation (Rohen, 1964; Hogan et al., 1971). Postnatally, in the irregularities on the surface of the inner limiting membrane fac ing the retina only a slight increase can be observed, which is determined by the growth of the retinal glia. 1.2. Nerve Fibre and Ganglion Cell Layer The layer of nerve fibres bordering on the inner surface of the retina contains the densely distributed axons of the ganglion nervi optici and the radial Miiller's fibres, which spread out to the inner limiting membrane like a fan, as well as the astrocytes and the inner retinal blood vessels. The next layer of ganglion cells outwards is characterized by the large, bright type II neurons of the visual pathway (Hogan et aI., 1971). In the cat, the ganglion cells outside the area centralis are arranged in one layer (Donovan, 1966). In the newborn cat, ganglion cells and delicate nerve fibre bundles are situated at about the same level, immediately below the inner limiting membrane. The axons, which are slightly differentiated (diameter about 0.2 -0.8 /.lm) are loosely packed to- 10

Postnatal Development of the Cat’s Retina

Similar Postnatal Development of the Cat’s Retina