Relation Between Superficial Capillaries and Foveal Structures in the Human Retina

Transcription

1 Relation Between Superficial Capillaries and Foveal Structures in the Human Retina Masayuki Iwasaki and Hajime Inomara When examining semithin Epon sections of human retinas, it became evident that superficial capillaries showed four different positions, according to the thickness of the ganglion cell layer. For the clear view of the distribution of the position of superficial capillaries, the intrafoveal region was subdivided into four zones, based on the thickness of the ganglion cell layer; the foveola and the A-, B-, and C-zone. The foveola has no ganglion cell layer, and the A-zone has a ganglion cell layer thinner than 15 nm. These regions lack superficial capillaries. In the B-zone, the ganglion cell layer is fim thick, and here the superficial capillaries lie in the outer boundary of the ganglion cell layer. The C-zone and parafovea have a ganglion cell layer thicker than 45 j*m, and superficial capillaries are present within the ganglion cell layer. The perifovea has a ganglion cell layer nm thick. In the temporal perifovea, where the nerve fiber layer is not so distinct, superficial capillaries are located on the outer boundary of the ganglion cell layer. In the other portion of the perifovea, superficial capillaries lie in the inner boundary of the ganglion cell layer. Out of the perifovea, where the ganglion cell layer is thinner than 15 nm, most of superficial capillaries touch both boundaries of the ganglion cell layer. Major retinal vessels touch the ganglion cell layer and lie in the similar position to that of superficial capillaries. Invest Ophthalmol Vis Sci 27: , 1986 Vascular patterns in the human retina have been studied mainly using whole mount preparations: trypsin digestion, 1 " 3 ink injection, 4 " 7 bentidine stain, 8 and plastic cast. 910 These studies clarified the laminated structure, that is, radial peripapillary, superficial, middle, and deep capillaries. Michaelson 8 found that major retinal vessels and superficial capillaries lay in the deeper level of the nerve fiber layer. On the other hand, Henkind 6 stated that major retinal vessels and superficial capillaries were located in the outer part of the nerve fiber layer, or within the ganglion cell layer. Thus, the position of the superficial capillaries is not so understood. One reason is the technical difficulty in observing retinal architectures in whole mount preparations. Another possible reason is that the central retina has further regional varieties in the ganglion cell and nervefiberlayer than in the inner plexiform and inner nuclear layers. The sectioning method may be the most suitable to observe the relation between retinal capillaries and the retinal architecture. Paraffin sections are, however, too thick, and the cytoplasm too poorly fixed, to clarify the exact relation. We examined semithin Epon sections of human retinasfixedfor electron microscopy, and found that the From the Department of Ophthalmology, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Submitted for publication: February 3, Reprint requests: Masayuki Iwasaki, MS, Department of Ophthalmology, Faculty of Medicine, Kyushu University 60, Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Japan. superficial capillaries had four different positions, according to the thickness of the ganglion cell and nerve fiber layer. Materials and Methods We examined intact retinas from four patients (43-65 yr of age) with carcinoma in a maxillary sinus, conjunctiva, and/or orbit. The enucleated eyes were immediately prefixated in 4% glutaraldehyde buffered at ph 7.2 with 0.1 M cacodylate. Small pieces of retinal tissues with choroid and sclerae were trimmed using a razor blade, taking care to avoid damage. Each tissue was mapped on a schema of the fundus, recording its distance and direction from the foveal center and/or the optic disc. Following several washings with fresh buffer solutions, the tissues were postfixated by 1% OsO 4 in the same buffer solution. After dehydration in a graded series ethanol and clearing with propylene oxide, they were embedded in Epon 812. Semithin sections 1 /xm thick were cut on a Sorvall MT-2B ultramicrotome (Du Pont; Newtown, CT) and stained for 1 min in 0.1% Azur-II with 1% borax at 90 C. Using about 30 retinal tissues, the position of superficial capillaries was observed light microscopically, and the thickness of each retinal layer was measured. Nomenclature Polyak 1 ' subdivided the central retina into three regions, "fovea," "parafovea," and "perifovea." The margin of the fovea is a point where the retina attains 1698

2 No. 12 CAPILLARIES AND THE FOVEA IN HUMAN RETINA / IwasoW and Inomoro 1699 its greatest thickness. Parafovea is the intermediate belt where the ganglion cell layer is composed of more than five rows of cells. Perifovea is the outermost region where the ganglion cell layer contains two to four rows of the cells. The area termed "foveola" did not have a margin which could be clearly distinguished, histologically. The mean thickness of the ganglion cell layer where the five rows are reduced to four, and from double rows into single, was about 45 pm and 15 nm, respectively. In the present study, we used the thickness of the ganglion cell layer instead of the number of rows of the cells to distinguish margins of the retinal regions. Thus, we could also subdivide the intrafovea into four zones from the center toward the foveal margin, foveola, and A-, B-, and C-zone. The retinal region without the ganglion cell layer is referred to as a foveola. A concentric zone around the foveola with the ganglion cell layer thinner than 15 fim is referred to as a A-zone. An intrafoveal zone where the ganglion cell layer is /im in thickness is termed the B-zone. An intrafoveal zone with a ganglion cell layer thicker than 45 fim is referred to as the C- zone. The margin of the fovea is distinguished by the greatest thickness of the ganglion cell layer. A zone surrounding the fovea with the ganglion cell layer central retina Thickness of ganglion cell layer Fig. 1. Relationship between retinal regions and the thickness of the ganglion cell layer. The upper right graph is a spline curve drawn by a microcomputer, on the basis of mean values from our measurements, a = A-zone, b = B-zone, c = C-zone, d = parafovea, e = perifovea. thicker than 45 /im is referred to as a parafovea. An extrafoveal zone where the ganglion cell layer is /zm thick is called the perifovea. Therefore, ganglion Fig. 2. A light micrograph of a flat section of the retina in a region of 5 mm superior from the optic disc. The sectioning plane is at the level of superficial capillaries. Most of the superficial capillaries are longitudinally sectioned in this plane. (X560)

3 1700 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1986 Vol. 27 o o 100pm 100pm QCL ONL RPE QCL ONL RPE Fig. 3. Traces of vertically sectioned retinas temporal (a) and nasal (b) from the foveal center. Each contains a foveola (left), A-zone, B-zone, and C-zone (right). A,, and indicate capillaries which touch the inner, outer, and no boundary, respectively. Superficial capillaries are absent in the foveola and A- zone. In the B-zone, superficial capillaries are present in the outer boundary of the ganglion cell layer. In the C- zone, they are present within the ganglion cell layer. Shifting point of the position of superficial capillaries in a is farther from the foveolar margin than that in b. The thickness of the ganglion cell layer, however, is much the same, because increase in the ganglion cell layer thickness in a is slower than that in b. Fig. 4. A light micrograph of a vertically sectioned retina, which contains a foveola (left), A-zone, and B-zone (right). The superficial capillary nearest to the foveal center is seen in the upper right. (X56O)

4 No. 12 CAPILLARIES AND THE FOVEA IN HUMAN RETINA / Iwosoki and Inomoro 1701 Fig. 5. The inner layers of a vertically sectioned retina, a, B-zone. The ganglion cell layer is moderately thick. Superficial capillaries are present in the outer boundary of the ganglion cell layer, b, C-zone. The ganglion cell layer is extremely thick. Superficial capillaries are present within the ganglion cell layer. Scale bar: thickness of the ganglion cell layer. (X560) cell layers of pairs of retinal regions on opposite sides of the foveal margin; A-zone and peripheral retina; B- zone and perifovea; and C-zone and parafovea; have the same thickness (Fig. 1). Results Extent of Newly Subdivided Retinal Regions We obtained only three tissues which contained foveal centers and both of the foveolar margins. The diameter of the foveola was 0.3 mm and 0.7 mm horizontally, and 0.4 mm vertically. The peripheral margin of the A-zone, where the ganglion cell layer was 15 ^m thick, was 0.12 ± mm (n = 7) distant from the foveolar margin. The peripheral margin of the B-zone, where the ganglion cell layer was 45 /j.m thick, was 0.54 ±0.15 mm (n = 6) distant from the foveolar margin. The peripheral margin of the C-zone, where the ganglion cell layer had the maximal thickness of 58 ± 7.9 nm (n = 5), was 0.92 ±0.11 mm (n = 5) distant from the foveolar margin. The foveal margin in the conventional subdivision, an area where the total retina attained its greatest thickness of 274 ± 26 fim (n = 5), was 0.91 ± mm (n = 5) distant from the foveolar margin. It was almost the same as the peripheral margin of the C-zone.

5 1702 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1986 Vol. 27 GCL INL ONL ONL RPE RPE 100)jiti 100pm Fig. 6. Traces of retinas in the extrafoveal regions, a, Infero-temporal portion of parafovea (left) and perifovea (right). The nerve fiber layer is developed. In the parafovea, superficial capillaries are within the ganglion cell layer. In the perifovea, superficial capillaries are located in the inner boundary of the ganglion cell layer, b, Temporal portion of the parafovea (left) and perifovea (right). The nerve fiber layer is not distinct. In the parafovea, superficial capillaries are within the ganglion cell layer. In the perifovea, superficial capillaries are present in the outer boundary of the ganglion cell layer. The peripheral margins of the parafovea and perifovea, where the ganglion cell layers were 45 urn and 15 fim thick, were about 1.4 mm and 3 mm distant from the foveolar margin, respectively. These values are not exact, as they were obtained by adding two values from two neighboring tissues, since their margins were too distant from the foveolar margin. These results are summarized in Figure 1. Identification of Superficial Capillaries In a vertical section, the superficial capillary net was observed as a single row of isolated superficial capillaries sectioned transversely and, rarely, obliquely or longitudinally. In a flat section, however, most were cut longitudinally, and took the form of a single continuous tree-like net (Fig. 2). Vertical sections contained exceptionally positioned superficial capillaries in the ganglion cell layer, at a rate of about 10%. Serial sections revealed some of them to be vertical capillaries connecting the superficial capillary net with deeper nets, while others belonged to the superficial capillary net. In this study, we regarded, by a single vertical section, all capillaries making contact with the ganglion cell layer to be superficial ones. Position of Superficial Capillaries An intrafoveal region with a very thin ganglion cell layer had no capillaries. The capillary nearest to the foveal center was a superficial one situated 0.15 ± mm (n = 7) distant from the foveolar margin, where the ganglion cell layer was 16.7 ±3.4 /xm (n = 7) thick. This avascular zone was all but equal to the foveola and A-zone (Figs. 3, 4). In an intrafoveal region where the ganglion cell layer had a moderate thickness, superficial capillaries were evident in the outer (or scleral) boundary of the ganglion cell layer (Figs. 3, 5a). A region near the foveal margin with an extremely thick ganglion cell layer had superficial capillaries within the ganglion cell layer (Figs. 3, 5b). The shifting point of the superficial capillaries from the outer boundary to the inside of the ganglion cell layer was 0.46 ± 0.21 mm (n = 6) distant from the foveolar margin, which had a marked variation. The thickness of that point was, however, rather constant at 40 ± 6.3 nm (n = 6). The concentric zone where the superficial capillaries lie in the outer boundary was practically equal to the B-zone. In the extrafoveal region where the ganglion cell and nerve fiber layer were of a moderate thickness, superficial capillaries were observed in the inner (or vitreal) boundary of the ganglion cell layer (Figs. 6a, 7a). The temporal portion, where the ganglion cell layer was of a moderate thickness but the nerve fiber layer was not so distinct, superficial capillaries were present in the outer boundary of the ganglion cell layer (Figs. 6b, 7b). The point where the superficial capillaries shifted from the inside to the inner or outer boundary was about 1.3 mm distant from the foveolar margin, and the thickness of the ganglion cell layer was approximately 45 /xm. This region, where the superficial capillaries existed within the ganglion cell layer, was practically equal to the C-zone and parafovea. The extrafoveal region, where the superficial capillaries were in the inner or outer boundary, corresponded to the perifovea. Out of the perifovea, the ganglion cell layer gradually grew thinner with distance. The thinner the ganglion cell layer, the more frequently the superficial capillaries came into contact with both the inner and outer boundaries of the ganglion cell layer (Fig. 8). These observations are summarized schematically in Figure 9. Position of Major Retinal Vessels Most of the major retinal vessels touched the ganglion cell layer and lay in a similar position to that of the neighboring superficial capillaries. The larger the vessels, the greater the tendency to contact both

6 No. 12 CAPILLARIES AND THE FOVEA IN HUMAN RETINA / Iwasaki and Inomoro w %?#; 35j.im Fig. 7. Light micrographs of the inner layers of the perifoveal retinas. The ganglion cell layer is of a moderate thickness, a, Superior portion. The nerve fiber layer is well developed. Superficial capillaries are present in the inner boundary of the ganglion cell layer. A large vessel (superotemporal venule) touches both the boundaries of the ganglion cell layer. Radial peripapillary capillaries are seen in the nerve fiber layer. b? Temporal portion. The nerve fiber layer is not distinct. Superficial capillaries are present in the outer boundary of the ganglion cell layer. Scale bar = thickness of the ganglion cell layer. (X560) boundaries (Figs. 7, 8). Therefore, these major branches and superficial capillaries form a single superficial vascular net. Discussion The subdivision of the intrafoveal region determined in the present work was based on the thickness of the ganglion cell layer. This represents not only the retinal architecture, but also the distribution of the position of the superficial capillaries. We noted four different positions of the superficial capillaries. Most portions of the perifovea in which superficial capillaries were present in the inner boundary of the ganglion cell layer were of the largest extent in

7 1704 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / December 1986 Vol. 27 Fig. 8. The retina out of perifovea. The ganglion cell layer is very thin, and the nerve fiber layer is developed. Superficial capillaries touch both inner and outer boundaries of the ganglion cell layer. Scale bar = thickness of the ganglion cell layer. (X56O) the central retina. The retina in this region shows a typical architecture, consisting of 10 layers with a moderate thickness. We conclude that the inner boundary, which is convenient to nourish both the ganglion cell and nervefiberlayer, is the fundamental position of the superficial capillaries. The other positions are probably modified ones to adapt to the specialized retinal architecture. Superficial capillaries were present within the ganglion cell layer in the C-zone and parafovea, an area where the ganglion cell layer was thicker than 45 fxm. The eccentricity of the point where superficial capillaries shifted from the outer or inner boundary to the inside had a marked variation, while the thickness of the ganglion cell layer, at that point, was rather constant. This indicates that the most important factor for Fig. 9. A schema of distribution of the position of superficial capillaries. D = region where superficial capillaries are absent, H = region where superficial capillaries are in the inner boundary of the ganglion cell layer, H = region where superficial capillaries are in the outer boundary of the ganglion cell layer, S3 = region where superficial capillaries are within the ganglion cell layer, Q = region where superficial capillaries touch both boundaries of the ganglion cell layer, a = A-zone, b = B-zone, c = C-zone, d = parafovea, e = perifovea. central retina

8 No. 12 CAPILLARIES AND THE FOVEA IN HUMAN RETINA / Iwosoki and Inomara 1705 determination of the position of superficial capillaries is the thickness of the ganglion cell layer. It is suggested that the superficial capillaries shift to the inside to nourish the ganglion cells located more than 45 /xm from the inner boundary. Therefore, the 45 ^m is probably a maximal depth for the superficial capillaries to be able to nourish ganglion cells. In Michaelson's measurement 8 of the mesh of the superficial capillary net, the average width of the mesh was 65 jim in 9-10 mm lateral from the optic disc, and 100 /xm in mm lateral. Half of these values should be the maximal distance for superficial capillaries to provide nourishment. It is of interest that the value of the maximal distance roughly coincides with our assessed value of the maximal depth. Superficial capillaries exist in the outer boundary of the ganglion cell layer in the B-zone and temporal perifovea. Thus, there is probably no need for the superficial capillaries to exist in fundamental positions, because the nerve fiber layer is extremely thin. This indicates that the second factor to determine the position of superficial capillaries is the thickness of the nerve fiber layer. From the present study, we conclude that superficial capillaries mainly nourish the ganglion cell layer, then the nerve fiber layer and other inner layers, additionally. Their fundamental position is the inner boundary of the ganglion cell layer, and they have three modified positions, according to specialization of the retinal architecture. Key words: capillary, fovea, human retina, ganglion cell layer, light microscopy References 1. Kuwabara T and Cogan DG: Studies of retinal vascular patterns. Part I. Normal architecture. Arch Ophthalmol 64:904, Toussaint D, Kuwabara T, and Cogan DG: Retinal vascular patterns. Part II. Human retinal vessels studied in three dimensions. Arch Ophthalmol 65:575, Cogan DG, Toussaint D, and Kuwabara T: Retinal vascular patterns. Part IV. Diabetic retinopathy. Arch Ophthalmol 66:366, Michaelson IC and Steedman HF: Injection of the retinal vascular system in enucleated eyes. Br J Ophthalmol 33:376, Ashton N: Injection of the retinal vascular system in the enucleated eye in diabetic retinopathy. Br J Ophthalmol 34:38, Henkind P: New observations on the radial peripapillary capillaries. Invest Ophthalmol 6:103, 1967a. 7. Henkind P: Radial peripapillary capillaries of the retina. I. Anatomy: human and comparative. BrJ Ophthalmol 51:115, 1967b. 8. Michaelson IC: Retinal Circulation in Man and Animals. Springfield, Charles C Thomas, 1954, pp Wybar KC: Anastomoses between the retinal and ciliary arterial circulations. Br J Ophthalmol 40:65, Shimizu K and Ujiie K: Structure of Ocular Vessels. Tokyo, Igaku-Shoin, 1978, pp Polyak SL: The Retina. Chicago, University of Chicago Press, 1941, pp