Investigative Ophthalmology & Visual Science, Vol. 29, No. 6, June 1988 Copyright © Association for Research in Vision and Ophthalmology Selective Loss of Retinal Ganglion Cells in Albino Avian Glaucoma Koichi Takatsuji,* Masaya Tohyama,t Yoshio 5ato4 and Akira Nakamura§ Retinal ganglion cell loss was investigated in the retinae of albino quails before and after the develop- ment of glaucoma. The isodensity maps of ganglion cells, the total number of ganglion cells, and the histograms of the cell size in the central region of the retina were similar between albino quails without glaucoma and pigmented quails. However, ganglion cells in the intermediate and peripheral regions of the albino quail retina without glaucoma were significantly smaller than those of the pigmented quail retina. In albino quails with moderate glaucoma in 3 months of age, 11% to 55% of all the retinal ganglion cells had disappeared, with the loss of medium-sized cells (30-60 urn2) occurring earlier than that of small and large cells. In albino quails with advanced glaucoma, there was marked cupping around the optic nerve head, and only small ganglion cells remained in the ganglion cell layer. Invest Ophthalmol Vis Sci 29:901-909,1988 We found imperfect albino mutant quails with a coma, paying particular attention to the ganglion sex-linked recessive gene.12 These quails have white cells most affected. feathers except on their back, and ruby-colored eyes instead of brown. There are few pigment granules in Materials and Methods the pigment epithelium, choroid and pecten oculi. Albino mutant quails (Coturnix cotumix japonica, Some pigment granules, however, were noted in the gene symbol at) at 3 (n = 11) and 6 (n = 8) months of ora serrata, ciliary processes and iris.3 After the age of age, and 6-month-old pigmented quails (n = 5) were 3 months, these quails develop closed-angle glau- used. They weighed about 100 g. Their breeding was coma, which is associated with reduced corneal cur- 4 5 described previously. ' Lenses were examined with a vature and lens intumescence.4'5 In glaucoma, retinal binocular slit lamp microscope to observe the extent ganglion cells and their fibers degenerate and disap- of opacity. The iridocorneal angles were examined pear. Two mechanisms leading to the degeneration of with a Koeppe-type gonioprism modified for small retinal ganglion cells have been proposed: mechani- animal eyes. cal alterations and vascular insufficiency arising from elevated intraocular pressure.6'7 Visual field loss is The quails were anesthetized with sodium pento- caused by defects in retinal ganglion cells in glau- barbital (2.5 mg/100 g, i.p.). After the intraocular pressure (IOP) was measured by the cannulation coma patients. However, little attention has been 5 paid to ganglion cell loss of the retina in experimental method, the quails were immediately killed by or clinical glaucoma. In the present study, we first transcardiac perfusion of 2% glutaraldehyde-2% para- compared the cell density, cell size, and total number formaldehyde in a 0.1 M phosphate buffer at pH 7.4. of ganglion cells between albino quails before the de- Lens, cornea and sclera were removed from each eye, velopment of glaucoma and pigmented quails to clar- and the remaining eye cup was fixed in the same fresh ify whether there was congenital retinal ganglion cell fixative for 24 hr. Then the pecten and optic nerve loss in the albino quail. Then we examined the pro- head were cut at their base, to determine their orien- cess of ganglion cell loss in albino quails with glau- tation. The retina was carefully dissected from the underlying pigment epithelium and soaked in 0.1 M phosphate buffer at pH 7.4. The isolated retina was mounted on a slide with 0.75% gelatin in water. A From the *Osaka Prefectural College of Nursing, Osaka, the Departments of fAnatomy and ^Common Laboratory, Osaka fine brush was used to spread the retina, vitreous sur- University Medical School, Osaka, and the §Shizuoka Women's face up, to obtain maximum contact with the gelatin. College, Hamamatsu, Japan. The slide was air-dried overnight at room tempera- Submitted for publication: December 23, 1986; accepted Jan- ture. The retina was stained with 0.2% cresyl violet uary 21, 1988. for 60 min. The slide was dehydrated in increasing Reprint requests: Koichi Takatsuji, DMSc, Osaka Prefectural concentrations of alcohol, cleaned in xyline and cov- College of Nursing, Tezukayamahigashi 2, Sumiyoshiku, Osaka 558,Japan. erslipped using Permount 0 (Fisher Scientific Co., 901 Downloaded from iovs.arvojournals.org on 09/26/2021 902 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1988 Vol. 29 NJ). The outline of whole-mount retina was traced by projection onto the easel of a photographic enlarger. The retinal area up to the ora serrata was measured twice using an image analyzer (Mutoh Digigrammer Model G linked to a Canon BX1 computer, Tokyo, Japan), immediately after mounting on the slide and coverslipping. Estimated shrinkage was from 1.0% to 3.8%. Shrinkage was so slight that no correction was made for measurement of the size of retinal ganglion cells. In 33 retinae, the number of cells in a 75 ^.m2 field was counted at a 1000-fold magnification. About 140 sample areas were assessed for each retina, and iso- Fig. 1. Light micrograph of the ganglion cell layer in the periph- density lines based on cell number were made. The eral region of an albino quail retina. Many ganglion cells and glial total number of ganglion cells in each retina was esti- cells (arrowheads) are seen. Cresyl violet stain. Bar, 25 /urn. mated from the mean neuron density between pairs Fig. 2. lsodensity maps of cells in the ganglion cell layer of retinae. (A) Pig- mented quail in 2R, (B) al- bino quail without glau- coma in 9R, (C) and (D) al- bino quails with moderate glaucoma in 17R and 26R, (E) albino quails with ad- vanced glaucoma in 22R. Numerals on the lines rep- resent 1000 cells/mm2. Note lower cell density at the dorsotemporal part in D. The optic nerve head is dotted. N, nasal; T, tem- poral; V, ventral. Bar, 5 mm. Downloaded from iovs.arvojournals.org on 09/26/2021 No. 6 SELECTIVE GANGLION CELL LOSS IN AVIAN GLAUCOMA / Tokorsuji er ol. 903 of isodensity lines multiplied by the intervening area. Table 1. Total number of neurons in the ganglion In some regions of the retina magnified 1000 times, cell layer and retinal area of pigmented quails, the ganglion cells in the ganglion cell layer were and of albino quails traced using a drawing tube, and the cell size was Neurons Retinal area measured with an image analyzer. Animal care and Animals (X104) (mm2) treatment in this investigation were in compliance with the ARVO Resolution on the Use of Animals in Pigmented quails 1 L 224 164 Research. 2R 261 170 3R 268 163 Results 4L 263 163 5L 248 168 Observation of Quail Eyes Mean ± SD 253 ± 16 166 ±3 In pigmented quails, the IOP was less than 18 mm Hg, the iridocorneal angle was open and the lens was Albino quails without 3 glaucoma clear. Similar findings were observed in albino quails 9R 260 172 at 3 months of age. Since albino quails showed wide 13R 265 180 variation in the time of onset of glaucoma, they were 14 L 244 167 15 L 258 160 divided into two groups, according to their IOP. One 33 L 232 165 group consisted of albino quails with moderate glau- Means ± SD 252 ± 12 169 ±6 coma (IOP between 18 and 23 mm Hg), in which the Albino quails with lens was slightly opaque in the anterior and posterior moderate cortical regions, and the iridocorneal angles in most glaucoma of them were open, but narrow. The other group 10 L 198 210 consisted of those with advanced glaucoma (IOP 11 L 217 183 11 R 146 177 higher than 24 mm Hg), in which the entire lens was 16 L 208 178 opaque, the iridocorneal angle was closed, and pupil 17 L 145 177 17R 175 180 reflex to the light was absent. When the cornea and 20 L 198 190 lens were removed from the eyes, marked cupping 20 R 191 191 around the optic nerve head was seen. 21 L 115 185 23 L 205 187 26 R 164 175 Isodensity Maps of Ganglion Cells on 27 L 173 187 27 R 161 189 Whole-Mount Retina 32 R 228 182 Figure 1 shows the ganglion cell layer in the periph- 34 R 235 178 ery of an albino quail retina. Two types of cells were Mean ± SD 185 ±25 186 ± 11 observed, neuron and glia. Neurons were over 4 /tm Albino quails with in diameter, round and rich in Nissl substance, which advanced glaucoma we counted and measured. Glias were below 4 nm in 7 L 35 188 diameter and irregularly shaped. Both cell types were 7 R 37 185 present in the retinal ganglion cell layer of the pig- 21 R 33 186 22 L 29 170 mented quails. 22 R 34 165 The isodensity map of ganglion cells in the albino 24 L 43 170 quail without glaucoma was similar to that in the 24 R 35 175 pigmented quail (Fig. 2A, B). In these two groups, the 26 L 58 179 cell density (more than 35,000 cells/mm2) was high- Mean ± SD 33 ± 18 185 ±8 est in the dorsotemporal region to the optic nerve Individual animal indicated in figures. L: left eye, R: right eye. head, and tended to decrease toward a periphery (about 10,000 cells/mm2).