Development of Microcystoid Macular Degeneration in the Retina of Nonhuman Primates: Time-Course and Associated Pathologies

Abstract

Purpose: Microcystoid macular degeneration (MMD) is a condition where cystoid vacuoles develop within the inner nuclear layer of the retina in humans in a variety of disorders. Here we report the occurrence of MMD in non-human primates (NHPs) with various retinal ganglion cell (RGC) pathologies and evaluate the hypothesis that MMD does not precede RGC loss but follows it.

Methods: Morphological studies were performed of the retinas of NHPs, specifically both rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis), in which MMD was identified after induction of experimental glaucoma (EG), hemiretinal endodiathermy axotomy (HEA), and spontaneous idiopathic bilateral optic atrophy. In vivo imaging analyses included fundus photography, fluorescein angiography (FA), optical coherence tomography (OCT), adaptive optics scanning laser ophthalmoscopy (AOSLO), light microscopy, and electron microscopy.

Results: MMD, like that seen on OCT scans of humans, was found in both rhesus and cynomolgus macaques with EG. Of 13 cynomolgus macaques with chronic EG imaged once with OCT six of 13 animals were noted to have MMD. MMD was also evident in a cynomolgus macaque with bilateral optic atrophy. Following HEA, MMD did not develop until at least 2 weeks following the RNFL loss.

Conclusion: These data suggest that MMD may be caused by a retrograde trans-synaptic process related to RGC loss. MMD is not associated with inflammation, nor would it be an independent indicator of drug toxicity per se in pre-clinical regulatory studies. Because of its inconsistent appearance and late development, MMD has limited use as a clinical biomarker.

Keywords: Microcystic macular degeneration; endodiathermy axotomy; experimental glaucoma; inner nuclear layer; optic atrophy.

Figure 1.

Visual field (left frame) and vertical OCT scan (right frame) of a 72-yearold woman who suffered an inferior hemiretinal branch vein occlusion several years earlier. Microcystoid spaces (arrows) in the inner nuclear layer, greatly thinned nerve fiber layer, and ganglion cell layer are confined to the inferior retina. Gray arrow points to the normal superior nerve fiber layer.

Figure 2.

A cynomolgus monkey (Animal #1, Table 1) with end-stage experimental glaucoma (EG) in the right eye of several years’ duration (frames; B, D, F) compared to the fellow control left eye (frames; A, C, E). B: Pale optic nerve with subtotal cupping is evident on the color fundus images of the right eye. C and D: Horizontal OCT scans through the fovea (corresponding to white scan direction lines in the color images) show normal retinal layers in the left eye (C) but MMD in the right eye (D, arrows). E and F: En face near infrared scans of the fellow control left eye (E) and the experimentally glaucomatous right eye (F). The fovea appears white in the fellow control left eye (E) due to segmentation errors. The experimentally glaucomatous right eye has a classic doughnut pattern of MMD surrounding the uninvolved fovea (F). G and H: AOSLO scans of the inferotemporal retina at the level of the inner nuclear layer of a rhesus monkey with EG. Distinct microcysts are present in the experimentally glaucomatous eye (H), which are medium to dark gray (hyporeflective) and surrounded by a thin white (hyperreflective) border (yellow asterisks denote three of the dozens of microcysts that occupy almost all this frame).

Figure 3.

Transmission electron micrographs of the same cynomolgus monkey shown in Figure 2 with end-stage EG. Left frame: retina of fellow control left eye showing the inner plexiform layer (IPL), outer nuclear layer, and outer plexiform layer (OPL). Right frame: Right eye with experimental glaucoma. A large microcystoid cavity (M) is evident. Note that this space is not epithelial lined. In addition, smaller cystoid spaces are present, some of which appear to be intranuclear (arrows) but are most likely nuclear indentations. The separation between the IPL and OPL is greater in the EG eye due to the cystoid spaces in the outer nuclear layer. Bar = 10 µm.

Figure 4.

A 3-year-old female cynomolgus monkey that was found to be behaviorally blind had advanced optic atrophy of unknown etiology in both eyes. The intraocular pressures were normal. Left frame: Fundus autofluorescence of the left eye. A darker, doughnut-shaped area is present in the macula that spares the fovea. The long white arrow indicates the level of the horizontal OCT scan shown in the right frame. The pattern is like that seen in the en face OCT scan of an experimentally glaucomatous monkey shown in Figure 2F. Right frame: Horizontal OCT scan through the fovea. MMD is indicated by the short white arrows. The retinal nerve fiber layer (RNFL) is absent (see Figure 5 white arrows for RNFL comparison), consistent with optic atrophy.

Figure 5.

Composite of cynomolgus and rhesus monkeys that had undergone hemiretinal endodiathermy axotomy (HEA). A: Fundus photograph of the right eye of a cynomolgus monkey taken immediately after endodiathermy had been applied adjacent to the inferior 180^o of the peripapillary area. B: Late phase fluorescein angiogram of a rhesus monkey that had undergone HEA 137 days prior. Unlike cystoid macular edema due to uveitis, there is no late fluorescein leakage in the macula. C: Vertical OCT scan through the fovea of same eye as shown in frame B prior to HEA. White arrows point to intact RNFL. D: Vertical OCT scan from the same eye as shown in frame C 137 days following HEA to the inferior peripapillary area. The RNFL from the superior retina is unaffected by HEA (white arrow) but absent from the inferior retina (gray arrow). MMD is confined to the inferior, axotomized hemiretina (black arrow). E: Paraphenylenediamine staining of a cross section of the optic nerve of the right eye shown in frame A three months after HEA. There is normal dark myelin staining of the superior optic nerve but absence of staining inferiorly. Bar = 250 µm. F: Light micrograph (toluidine blue) of the inferior (axotomized) hemiretina of another cynomolgus monkey showing the MMD spaces in the inner nuclear layer, absent RNFL, and greatly decreased retinal ganglion cell layer. Bar = 50 µm.

Figure 6.

Sequential vertical OCT scans through the retina of a rhesus monkey that had undergone HEA to the inferior peripapillary area. The RNFL of the inferior retina is evident at baseline and at Week 1 (asterisks), is greatly decreased at Week 2, and is absent at subsequent weeks. At Week 4, the RNFL is absent, but MMD (arrows) is not visible and does not appear until Week 8. MMD is still present at Week 12.

Figure 7.

Left graph: Mean RNFL thickness over time following inferior retina HEA in four monkeys. Week “0” indicates baseline measurements. The RNFL thickness in the superior retina is unaffected. However, the RNFL thickness in the inferior retina is markedly decreased by Week 4. Error bars are standard errors of the mean. Right graph: Mean inner nuclear layer (INL) thickness over time following inferior retina HEA in four monkeys. Week “0” indicates baseline measurements. The INL thickness in the superior retina is unaffected. However, there is a trending increase in INL thickness of the inferior retina by Weeks 8 and 12. The increasing size of the error bars of the later weeks is consistent with the variable presence of MMD, unlike the monotonic decrease in RNFL thickness after Week 1 post HEA.