FULL PAPER Anatomy Relationship between Distribution of Tapetum Fibrosum and Retinal Pigment Epithelium in the Sheep Eye Aya SHINOZAKI1,2), Yoshinao HOSAKA2), Tomohiro IMAGAWA2) and Masato UEHARA2)* 1)The United Graduate School of Veterinary Science, Yamaguchi University, 1677–1 Yoshida, Yamaguchi 753–8515 and 2)Department of Veterinary Medicine, Tottori University, 4–101 Koyama Minami, Tottori 680–8553, Japan (Received 14 September 2009/Accepted 10 October 2009/Published online in J-STAGE 27 November 2009) ABSTRACT. The tapetum lucidum is a light-reflective device improving visual sensitivity in mesopic vision. There have been few studies on tapetal distribution and its relationship with degree of pigmentation in the retinal pigment epithelium (RPE). In the present study, sheep’s eyes were used for macroscopical observation of the tapetum, and then histological sections of the posterior eyecups were made to analyze the distribution of tapetal thickness and degree of pigmentation in the RPE. Macroscopically, with available light, the tapetum was located in the dorsal eye fundus to the optic disc and showed an L-shape with the horizontally elongated nasal part and the dorsally expanded temporal part. In photographs with a flash, the tapetal area expanded and showed a more triangular shape. The tapetum his- tologically consisted of layers of dense collagen fibers and was thicker in the temporal part than in the nasal part. The maximum tapetal thickness was approximately 70 m. The histological tapetal area was similar to the tapetal shape with a flash light. The pigmentation of the RPE was divided into three types, nonpigmented, transitional, and pigmented areas. The nonpigmented area was similar to the tapetal shape with available light. It is suggested that approximately 55% of the histological tapetal area is covered with the nonpig- mented area and is functional under a natural light condition. The functional tapetal area was similar to the L-shaped high-density area of retinal ganglion cells. KEY WORDS: area centralis, pigmentation, retinal ganglion cell, tapetum lucidum. J. Vet. Med. Sci. 72(2): 211–215, 2010 The tapetum lucidum is a light-reflective device found in ever, there have been few studies on the distribution of many vertebrates and invertebrates. The tapetum is consid- tapetal thickness, degree of pigmentation in the RPE, and ered to improve mesopic vision under a low light condition the relationship with the retina. Furthermore, the shape of by reflecting light that was not absorbed and passed through the sheep’s tapetum is somewhat different in some studies the photoreceptors and providing the photoreceptors with a and controversial [1, 6, 7, 13]. Thus, the objectives of the second chance for absorbing the reflected light [13, 14]. present study were to investigate the histological distribu- Most of the mammalian tapetum is the choroidal tapetum tion of tapetum tissues and the degrees of pigmentation in located outside the retina and between the choriocapillary the RPE on the tapetum throughout the sheep’s eye fundus layer and the proper substance of the choroid with many and to compare with distribution of retinal neurons. From melanocytes. The choroidal tapetum is divided into two the results, we are able to explain the difference in the types, the tapetum cellulosum and the tapetum fibrosum. sheep’s tapetal shape. To our knowledge, this is the first The tapetum cellulosum consists of layers of rectangular- report showing the topographic map of the tapetal thickness shaped tapetal cells containing numerous rodlets. The tape- and the degrees of pigmentation in the RPE throughout the tum fibrosum in sheep consists of closely and regularly eye fundus. arranged layers of collagen fibers, and the tapetum produces a structural metallic color in the sheep’s eye fundus [13, 14, MATERIALS AND METHODS 16]. The retinal pigment epithelium (RPE) is the outermost Right eyes from three adult Suffolk sheep (Ovis aries) layer of the retina, and the epithelial cells generally have (females, 4 years old, 74–83 kg) were used for the present numerous melanin granules. However, the RPE overlying study. The sheep were obtained after other experiments the tapetum has no melanin granules in order to allow light unrelated to those described here and killed by exsan- to arrive at the tapetum and to be reflected back to photore- guination under deep anesthesia by xylazine. All experi- ceptors [3, 5, 13, 14, 16]. ments were conducted in compliance with the guidelines of There have been many morphological studies on the tape- the Animal Care and Use Committee of Tottori University tum cellulosum using electron microscopy and some studies and the AVMA Guidelines on Euthanasia. on the tapetum fibrosum and the RPE [1–3, 5, 17]. The Each enucleated right eye was perfused with 2.5% glut- tapetum is considered to have some effects on vision, how- araldehyde-2% paraformaldehyde/0.1 M phosphate buffer (pH 7.4) into the eyeball in order to avoid separation of the *CORRESPONDENCE TO: UEHARA, M., Department of Veterinary Medicine, Tottori University, 4–101 Koyama Minami, Tottori retina and choroid from the sclera. After immersion in the 680–8553, Japan. same fixative for a few days, the eye was opened by an e-mail: [email protected] encircling cut, and the posterior eyecup was again fixed in 212 A. SHINOZAKI, Y. HOSAKA, T. IMAGAWA AND M. UEHARA Fig. 1. Photomicrographs of the tapetum and retinal pigment epithelium (RPE) in HE-stained vertical sections of a sheep eye. The tape- tum shows even and eosinophilic stainability. The RPE is variable in pigmentation. A: Thick tapetal area covered with nonpigmented RPE. B: Tapetal area covered with the transitional area of the RPE. C: Thin tapetal area covered with pigmented RPE. D: Nontapetal area covered with heavily pigmented RPE. The arrows with both heads show tapetal thickness. RPE=retinal pigment epithelium, CCL=choriocapillary layer, TL=tapetum lucidum, PSC=proper substance of choroid, BV=blood vessel, Sc=sclera. Scale bar =25 m. the same fixative. After fixation, the tapetal shape was RESULTS observed macroscopically and photographed with available light and with a flash. The posterior eyecup was divided Macroscopical observation of the tapetum (Fig. 2): Mac- into some parts in order to be flattened. Celloidin as embed- roscopically, with available light, the tapetum was located in ding medium was used to avoid separation of the retina and the dorsal eye fundus to the optic disc and showed an L- choroid, to accurately measure the tapetal thickness, and to shape consisting of the horizontally elongated nasal part and confirm the location of a section. Two serial vertical sec- the dorsally expanded temporal part (Fig. 2A). In photo- tions were made at 40–50 m in thickness every 1 mm or graphs with a flash, the tapetal area expanded, paticulally in every 200 m in a part including the area centralis of the ret- the dorso-central to dorso-nasal edge, and showed a more ina. The sections were stained with hematoxylin-eosin (HE) triangular shape (Fig. 2B). or trichrome stain with hematoxylin, acid fuchsin, and Histology of the tapetum and the RPE (Figs. 1 and 3): aniline blue. The specimens were pasted with a film includ- The tapetum was present between the choriocapillary layer ing lines at intervals of 1 mm. and the pigmented proper substance of the choroid. The Tapetal thicknesses were measured at 40 objective with tapetum consisted of many layers of dense collagen fibers HE specimens every 1 mm using an eyepiece with a scale including a few fibroblasts and a few small blood vessels attachment. The outer border of the tapetum was unclear penetrating the tapetal layers. The tapetum showed even because melanocytes were scattered in the outer border. and eosinophilic stainability in HE specimens and was blue Thus, the tapetal thicknesses were defined as the distance in color by aniline blue in trichrome staining. The outer from the inner point contacting the choriocapillary layer to tapetal edge was irregular due to irregular appearance of the outer point contacting melanocytes appearing in the melanocytes in the tapetal layers. tapetal layers (Fig. 1). The RPE was a single layer overlying the choriocapillary Degree of pigmentation in the RPE was observed at 20 layer. The RPE contained melanin granules of various objective between 1-mm-interval lines on the same HE amounts and sizes. In the thick tapetal area, few melanin specimens as those used for measurement of tapetal thick- granules were found in the RPE (Fig. 1A). In the thinner ness. RPE sampling areas were divided into three types by tapetal area, melanin granules increased gradually with degree of pigmentation. Firstly, the area in which nonpig- mented cells made up more than half of the RPE was regarded as the nonpigmented area with high light transmis- sion properties (Fig. 1A). In this area, there was little pig- mentation, if any. Secondly, the area in which pigmented cells made up more than half of the RPE was regarded as the transitional area with low light transmission properties (Fig. 1B). Thirdly, the area composed of only pigmented cells was regarded as the pigmented area with light impassable properties (Figs. 1C and 1D). From these data, topographic maps of tapetal thickness and degree of pigmentation in the RPE were made. The values were expressed as means SD. Fig. 2. Photographs of the sheep’s right eye fundus with avail- able light (A) and with a flash (B). The tapetal area shows an L- shape in A and a triangular shape in B. Right side is nasal. Scale bar =1 cm. TAPETUM FIBROSUM OF THE SHEEP 213 Distributions of tapetal thickness and nonpigmented area of the RPE (Fig. 4): The tapetal area histologically including tapetal layers (histological tapetal area) (Fig.