Foveal Ganglion Cell Loss Is Size Dependent in Experimental Glaucoma Yoseph Glovinsky,* Harry A. Quigley,~f and Mary E. Peasef Purpose. The purpose of this study was to study the pattern of foveal ganglion cell loss in experimental glaucoma. Methods. Retinal ganglion cell size and number in the foveal region of seven monkey eyes with experimental glaucoma was determined and compared to normal monkey eyes. Serial sections of macular retina were studied in two regions: the plateau of peak density of ganglion cells (800-1 100 M"I from the fovea), and within 500 /xm of the foveal center. Results. In normal eyes, cell densities were 37,900 ± 2700 in the foveal plateau and 17,200 ± 1800 cells/mm2 in the foveal center. There was selective loss of larger ganglion cells in glaucoma eyes. The degree of foveal ganglion cell loss was significantly correlated to the degree of nerve fiber loss in the temporal optic nerve of the same eye. Conclusions. Detection of early, central visual function loss in glaucoma could be enhanced by testing functions subserved by larger retinal ganglion cells. Invest Ophthalmol Vis Sci 1993; 34:395-400. .Large ganglion cells located in the retinal midperiph- cells, this information could help to select tests for ery are selectively damaged in human and experimen- early glaucomatous damage. We therefore studied the tal glaucoma.1'2 Glaucoma selectively decreases the ax- foveal area in eyes of monkeys with experimental glau- onal How to the magnocellular layers of the lateral coma and in normal eyes. geniculate body subserving the retinal midperiphery.3 Previous reports45 did not resolve whether there was MATERIALS AND METHODS size-dependent damage in the fovea. This is important because foveal function tests are abundant and simple This investigation adhered to the principles of the to use.6 If there is selective injury to foveal ganglion ARVO Resolution on Use of Animals in Research and was approved and monitored by the Institutional Ani- mal Care and Use Committee of the Johns Hopkins From the ^Glaucoma Service and Dana Center for Preventive University School of Medicine. Ophthalmology, Wilmer Ophthalmological. institute, Johns Hopkins Experimental glaucoma was induced in one eye of University School, of Medicine, Baltimore, Maryland, and the cynomolgus monkeys (Macaca fascicularis) by argon *Goldsch.leger Eye Institute, Sadder School of Medicine, Tel-Aviv 7 University, Tel-Aviv, Israel. laser applications to the trabecular meshwork. After Supported in part by PHS Research Grants EY 02120 and 01765 several months of intraocular pressure elevation, (HA Q) and, unrestricted research support from. National Glaucoma various levels of glaucomatous optic nerve damage oc- Research, a program of the American. Health Assistance Foundation, Rochvillc, Maryland, and a grant from Mr. G. Sheba (YG). curred. The monkeys were then exsanguinated under Submitted for publication: December 24, 1991; accepted August 31, intravenous sodium pentobarbital anesthesia by per- 1992. fusion with cold saline followed by 4% paraformalde- Proprietary interest, category: N. Reprints requests: Harry A. Qui.gl.ey, Wilmer 120, Johns Hopkins hyde and 2% glutaraldehyde. (In two monkeys only 4% Hospital, 600 N. Wolfe, Baltimore, MD 21205. paraformaldehyde was used.) The eyes were enucle- Invcsligativc Ophthalmology & Visual Science, February 1993, Vol. 'A4, No. 2 Copyright © Association for Research in Vision and Ophthalmology 395 Downloaded from iovs.arvojournals.org on 09/27/2021 396 Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2 ated and immersed in the perfusion fluid for 24 hr. A cross-section of the retrolaminar optic nerve was post- fixed in 1% osmium tetroxide and embedded in epoxy resin. Each eye was bisected at the equator and a 4 X 4 mm retina-choroid-sclera block with the foveola in its center was excised and postfixed in 2% osmium te- troxide. It was then placed in a mixture of equal parts of aged (2 wk at room temperature) saturated para- phenylenediamine (PPD) solution and 0.2 mol/l phos- phate buffer for 2 days at room temperature. A mix of one part 95% ethanol to two parts saturated PPD was next, and by adding small volumes of 95% ethanol every 15 min, the tissue was allowed to dehydrate grad- ually. The tissue was then immersed in a saturated so- lution of PPD in ] 00% ethanol for 1 hr, and for an- other hour in a saturated solution of PPD in propylene oxide. An equal volume of embedding resin was then added and shaken until it was dissolved. After 24 hr, the propylene oxide evaporated and the tissue was then embedded in the remaining blackened resin. Beginning at the nasal side of the block, semithin sections were cut until the center of the fovea was reached. Then, 50 serial 1-^rn sections were collected. At this area, the profiles of those retinal ganglion cells containing nucleoli were outlined under XI000 mag- nification with a camera lucida and a planimeter (Fig. 1). Cell diameter was calculated from their perimeter FIGURE 1. Retinal ganglion cells profiles in the plateau of (this was directly measured using the "length" option peak density. With the PPD block staining, cell borders are of the instrument), assuming that cells were circular. easily identified. (Original magnification XI000.) Cells of each fourth section were counted to allow a space of one nucleolus diameter (1.96 jum) between not. be used. Percentage cell loss was calculated for sections to avoid sampling a cell more than once. Cell each subgroup (bin) of cell diameter and was corre- density was calculated from the number of profiles lated to cell diameter. Selective damage of larger cells counted, section thickness, and average nucleolus size would be denoted by significant, positive slope of cell by the Abercombie formula.8 Cell density and diame- loss compared to diameter by linear regression analy- ter frequency distribution were studied in two areas: sis11 (P < 0.05). A similar analysis assessed the relation- (1) 0 to 500 jinn from the foveolar center (subserving ship of fiber loss to fiber diameter in the temporal the foveolar cones9) and (2) 800 to 1100 jum from the optic nerve sections of the same eyes. 9 foveolar center (the plateau of peak density ). Results The coefficient of determination (R2) was used to from equidistant areas above and below the foveolar estimate the amount of total variability in cell or nerve center were averaged. fiber loss that was accounted for by its linear relation- One-micron sections of the retrolaminar optic ship to cell or fiber diameter.'l For example, a correla- nerves were stained with toluidine blue. The number tion coefficient (R) of 0.7 between cell size and cell loss and diameter distribution of the fibers in the temporal means that 49% (0.72) of the variability in cell loss is half of each nerve were measured by the Zeiss IBAS related to the cell size, whereas 51% of the variability is image analysis system (Carl Zeiss, Inc., Thornwood, influenced by other factors. NY), as previously described.10 Twelve monkey eyes were studied: four normal RESULTS eyes, seven glaucomatous eyes, and one ocular hyper- tensive eye. Table 1 summarizes the clinical data of the In one normal eye, we measured the diameter of the seven monkey glaucomatous eyes. nucleolus in many cell profiles from vertical sections In each glaucomatous eye, cell density and diame- using a planimeter. The average nucleolus diameter ter distribution were compared to a control eye. This was 2.14 ± 0.15 ^m. This diameter did not vary among was the normal fellow eye in four monkeys, the ocular areas sampled every 100 pm between 400 to 1400 /xm hypertensive fellow eye in one, and an extensively stud- from the foveal center (Fig. 2). A tissue block of 12 ied normal eye (M7) in two eyes whose fellow eye could X 25 X 70 jum was then studied in detail. Each cell Downloaded from iovs.arvojournals.org on 09/27/2021 Foveal Ganglion Studies 397 TABLE l. Clinical Data on the Monkey Glaucoma Eyes Duration* Percent Temporalf Monkey No. Mean IOP (±1 SD) (mo) Vertical C/D Ratio Nerve Fiber Loss M. 26 ±2 0.6 M, 49 ± 5 0.8 M, 36 ± 5 0.7 21 M4 32 ± 6 0.3 15 M5 35 ± 1 0.8 63 Mr, 38 ±6 1.0 88 M7 45 ± 8 1.0 * Duration of elevated IOI*. t Axonal loss in the temporal half of the optic nerve. X Technical problem during post-fixation did not allow accurate counting of these optic nerve specimens. C/D, Cup/disc. profile in serial 1 fim sections of this block were mea- The ganglion cell densities in one normal eye at sured. Cell profiles that contained nucleoli were signif- various eccentricities are shown in Figure 4. As previ- icantly larger than those that did not (P < 0.000, t-test, ously reported,9 the peak density occurs 750 to 1100 Fig. 3). Also, when profiles of the same cell were stud- firm from the foveal center. Cell diameter distribution ied, those containing nucleoli were the largest ones measured every 100 jim showed either a distinct sec- and generally were located in the center of the cell. We ond peak of large cells (Fig. 5) or a skew toward larger therefore used the diameter of a profile containing the cell diameters. nucleolus as the best estimate of the true size of a The ganglion cell densities in the foveal center and cell.1213 In profiles of the same cell, we noted that in the plateau of peak density for four normal monkey small chromatin pieces in the nucleus might be con- eyes are listed in Table 2. The coefficient of variation fused with the nucleolus.