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 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.

Introduction

MMD is a condition that affects the inner nuclear layer (INL) of the retina and is associated with thinning of the RNFL and development of cystoid vacuoles within the INL. ¹ MMD is well documented in humans, being first associated with multiple sclerosis (MS). ^1–5 Subsequent research has found that MMD is not exclusive to MS, ^{4, 6–9} and may also be associated with neuromyelitis optica, ^{1, 10–12} glaucoma, ^13–18 and several additional types of optic neuropathies (Figure 1). ^19–23 Initially, the mechanism by which MMD emerges was proposed to be disruption of the blood-retinal barrier; ⁵ however, the evidence showing that MMD is not specific to MS rules this out as a mechanism, and subsequent research has suggested that the mechanism may instead be trans-synaptic retrograde degeneration. ^{19, 24} The comorbidity of trans-synaptic retrograde degeneration in the retina with MS²⁵ lends support to this hypothesis, although it is not without controversy. ²⁰ Retinal traction associated with epiretinal membranes and cystoid macular edema (from inflammation) can produce lesions that can masquerade as MMD, but which have distinctive morphological and physiological characteristics. ^{16, 26, 27}

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.

When first discovered, MMD was thought to have the potential to be a biomarker for MS. ^{2, 4, 5} However, not only did the discovery of MMD’s nonspecificity preclude its use as a biomarker, but so did the finding that MMD occurrence is dynamic and can either resolve or emerge in different areas on the retina. ^{4, 28} Furthermore, because MMD most often manifests late in the progression of MS, ^{5, 7} even if it were specific to MS it would not be observable before other biomarkers could allow for a diagnosis. While it may be useful as a severity indicator for a given neuropathy, ⁵ the dynamic nature of its occurrence makes it less reliable.

Although extensively documented in humans, MMD has only sparse description in nonhuman primates. In most of the published literature in which MMD is identified it is only briefly mentioned in the results and not further expanded upon. ^{29, 30} We report the occurrence of MMD in nonhuman primates with EG, idiopathic bilateral optic atrophy, and axon loss via HEA.

Methods

All experimental methods and techniques in these studies adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Wisconsin-Madison’s or the Labcorp Preclinical Drug Development’s Animal Care and Use Committees.

Experimental glaucoma.

EG was induced in two groups of adult cynomolgus macaques. The first group (Table 1) consisted of 6 females in which EG was induced in one eye for the purpose of testing potential IOP lowering agents. They had EG induced from 2.7 to 9.3 years. No IOP lowering agents were administered during the 14 months prior to OCT scanning (the period for which IOP data were available). A second group of 7 males had EG induced in one eye for 1 to 2 years (Table 2). IOP lowering drugs were administered to this group only if the IOP was greater than 60 mmHg. For all 13 animals, 532-nm diode laser was used to scarify the trabecular meshwork (laser trabecular destruction, LTD) as previously described. ^31–33 Briefly, intramuscular (IM) ketamine hydrochloride and IM acepromazine were administered for anesthesia. After topical application of 0.5% proparacaine hydrochloride ophthalmic solution to the cornea of the right eye, a 532-nm diode laser and slit-lamp delivery system (OcuLight GL; Iridex Corp., Mountain View, CA, USA) were employed to deliver laser light through a Kaufman-Wallow single-mirror monkey gonioscopy contact lens (Ocular Instruments, Inc., Bellevue, WA, USA). Confluent intense (1.0 W, 0.5 sec, 75 µm) laser spots were delivered to the inferior 270^o of the trabecular meshwork of the right eye. Intraocular pressures (IOP) were checked at least weekly and up to two additional laser treatments were administered (always sparing the superior one clock hour of trabecular meshwork) until the pressures were consistently above 25 mmHg as measured by a hand-held tonometer (Tono-Pen^® Vet, Reichert™, Depew, NY).

Table 1.

Six female cynomolgus monkeys with end-stage experimental glaucoma produced by laser trabecular destruction (LTD) of their right eyes (OD). Mean IOPs are for the prior 14 months (earlier data not available). IOPs units are mmHg (torr). Four of the six animals had OCT changes consistent with MMD (asterisks). However, two did not have observable MMD on OCT. There is no obvious correlation between MMD and IOP at the time of OCT. All the right eyes had marked reductions in RNFL thickness compared to the fellow control left eyes (OS). RNFL values were derived from manually segmented circumpapillary RNFL scans.

Animal #	Age (years)	Years after LTD	Mean IOP OD	Mean IOP OS	IOP at Scan OD	IOP at Scan OS	RNFL OD (µm)	RNFL OS (µm)
1*	11.8	9.3	23 ± 2	19 ± 2	22	21	29	104
2*	12.2	9.0	44 ± 8	17 ± 1	50	18	57	104
3*	5.7	3.1	49 ± 6	22 ± 1	47	23	38	91
4	7.8	4.1	53 ± 6	28 ± 1	47	26	46	93
5*	6.0	3.1	50 ± 8	23 ± 2	48	23	42	105
6	6.6	2.7	60 ± 8	25 ± 1	46	26	36	100

Table 2.

Seven male cynomolgus monkeys with shorter duration (5.0 to 6.8 months) experimental glaucoma produced by laser trabecular destruction (LTD) of their right eyes (OD) for which the entire IOP history is known. IOPs units are mmHg (torr). Two of the seven animals had OCT changes consistent with MMD (asterisks). There is no obvious correlation of MMD with IOP at the time of OCT. All the right eyes had marked reductions in RNFL thickness compared to the fellow control left eyes (OS). RNFL values were derived from manually segmented circumpapillary RNFL scans. RNFL thickness data for the left fellow control eye are not available (NA) for animal number 8 because of anesthesia complications.

Animal #	Age (years)	Duration of EG (months)	Mean IOP OD	Mean IOP OS	IOP at Scan OD	IOP at Scan OS	RNFL OD (µm)	RNFL OS (µm)
7	9.9	5.0	59 ± 7	20 ± 2	59	22	53	88
8	11.4	5.0	56 ± 10	19 ± 2	32	20	54	NA
9	9.0	5.9	56 ± 9	17 ± 2	59	16	45	116
10*	9.4	5.6	56 ± 10	24 ± 1	60	24	39	110
11	10.7	6.3	62 ± 9	23 ± 1	53	26	46	86
12	11.0	6.8	42 ± 10	21 ± 3	32	20	62	92
13*	9.6	5.9	44 ± 5	17 ± 2	42	17	64	107

Spontaneous idiopathic optic atrophy.

A drug naïve three-year-old, spontaneously blind, female cynomolgus macaque underwent physical and ophthalmic examination, fundus photography, and OCT. The animal was observed to be behaviorally blind and had pale optic nerves. No cause for blindness was established. The IOP in this animal was within a normal range in both eyes.

Hemiretinal endodiathermy axotomy (HEA).

Three adult rhesus macaques, one female and two males, and one adult female cynomolgus macaque were used in a study examining the structural changes associated with HEA procedures. All monkeys underwent HEA as previously described. ^{30, 32} Briefly, the animals were preanesthetized with ketamine and then intubated and anesthetized with an oxygen/isoflurane mix. Topical 1% tropicamide and 2.5% phenylephrine hydrochloride ophthalmic solutions were administered to dilate the animals’ pupils. To maintain a stable position for the procedure the animal’s head was supported in a temporary holding device. Proparacaine hydrochloride (0.5% ophthalmic solution) was administered as a local anesthetic, and the eyelids were retracted using a wire speculum. Two 25-gauge cannulae were inserted 4 mm posterior to the corneal limbus in the 2 o’clock and 10 o’clock meridians through the conjunctiva and sclera using trocars. A fiber optic light was passed through one cannula, and a sharp-tipped endodiathermy probe was passed through the other. A flat contact lens was used to visualize the retina through an operating microscope. Contiguous endodiathermy spots were placed on the inferior 180° adjacent to the optic nerve margin with enough energy to cause retinal whitening. OCT scans were performed at baseline (pre-HEA) and at 1, 2, 4, 8, and 12 weeks following the HEA procedure. Vertical OCT line scans through the fovea were segmented manually. The thickest points of the inferior and superior RNFL and INL layers were recorded. IOP measurements were within a normal range for all animals at all timepoints.

Imaging.

EG and Optic Atrophy Studies.

All animals were imaged using a Heidelberg Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany) spectral domain OCT. High resolution line and volume scans through the macula and RNFL circle scans around the optic nerve were obtained. OCT scans were segmented using the Heidelberg software with manual correction. Either a Topcon 50DX fundus camera (Topcon Corp., Tokyo, Japan), equipped with a Nikon D7000 camera or a TRC 50EX fundus camera with an EOS 5D Mark 2 camera (Canon, Tokyo, Japan), was used to acquire digital color fundus images. Adaptive optics scanning laser ophthalmoscopy (AOSLO) was used to obtain images for some EG fundi using the Imagine Eyes rtx1™ (Orsay, France).

HEA Study.

OCT scans were performed at baseline (pre-HEA) and at 1, 2, 4, 8, and 12 weeks following the HEA procedure. Vertical OCT line scans through the fovea were segmented manually. The thickest points of the inferior and superior RNFL and INL layers were recorded. IOP measurements were within a normal range for all animals at all timepoints. Fundus photography and fluorescein angiography imaging were obtained using the TRC 50EX retinal camera and captured using an EOS 5D Mark 2 camera connected to the retinal camera. Sodium fluorescein (10%) was used for angiography.

Histology.

Retinal sections were fixed in Richardson’s solution, ³⁴ embedded in epon, sectioned 1 µm thick and stained with toluidine blue. Thin sections were then cut for transmission electron microscopy. Some optic nerves were fixed in 4% paraformaldehyde, embedded in epoxy resin (Durcupan, Fluka--Sigma-Aldrich, St. Louis, MO), and stained with paraphenylene diamine.

Results

Experimental glaucoma (EG).

In the first group of cynomolgus monkeys with long term (2.7 to 9.3 years) EG, MMD was observed by OCT in four out of six monkeys (Table 1). The monkeys were part of a cross-sectional single evaluation of a long-term study of the effects of various topical IOP lowering medications. No drug testing was done for at least 14 months prior to OCT scanning. All experimentally glaucomatous right eyes featured a marked reduction in the RNFL. IOP data were only available for the 14 months prior to OCT scanning. One of the four eyes featuring MMD also had an IOP in the normal range at the time of scanning. A second group of cynomolgus monkeys had EG induced from 5.0 to 6.8 months prior to OCT. MMD was observed in two out of seven animals (Table 2). As with the first group, there was no obvious correlation between MMD and average IOP or RNFL thickness. When present, the MMD evident on the OCT scans corresponded to the doughnut-shaped, foveal sparing defects seen on en face scanning (Figure 2F). Lacunae consistent with MMD in the INL were also seen using AOSLO in three rhesus monkeys with EG as part of another study (Figure 2G and 2H).

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).

Electron microscopy obtained in three of the MMD-positive monkeys in the first group showed numerous microcystoid spaces in the INL. The sizes of the spaces varied from a few microns to tens of microns and were always extracellular (Figure 3).

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.

Spontaneous idiopathic optic atrophy.

MMD was identified using OCT in a cynomolgus monkey reported to have spontaneous idiopathic optic atrophy (Figure 4). The pattern of the MMD was indistinguishable from that of advanced EG on both OCT scanning and en face scanning. In addition to marked RNFL thinning, microcystoid spaces were present in the INL of both eyes that were distributed in a doughnut pattern in the macula sparing the fovea. Unlike most of the EG animals, the IOPs were never found to be elevated in this monkey.

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.

Hemiretinal endodiathermy (HEA).

MMD was observed in both cynomolgus and rhesus monkeys following HEA (Figure 5). MMD only developed in the portion of the retina affected by the axonal loss (i.e., decreased RNFL on OCT) and it was not associated with late leakage on fluorescein angiography (Figure 5B). To test the hypothesis that MMD does not precede RNFL loss but follows it, four monkeys (three rhesus and one cynomolgus) underwent HEA and the superior and inferior RNFL and INL thicknesses were compared. Subjective inspection of the line scans over time showed loss of inferior RNFL evident by Week 2 but MMD was not readily apparent on the OCT scans until Week 8 (Figure 6). Figure 7 (left graph) illustrates this effect graphically. Segmentation of the INL (which is the location of the MMD) showed an increase in INL thickness but was much more variable than the decrease in RNFL thickness following HEA (Figure 7, right graph).

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.

Discussion

MMD is found in both cynomolgus and rhesus macaques in a variety of conditions, such as EG, spontaneous idiopathic optic atrophy and HEA—all of which are associated with marked loss of the retinal ganglion cells and their axons. MMD thus appears to share a common etiology and may co-occur with a variety of disorders that damage the optic nerve in the human eye. ³⁵

Although prior publications have used the term “microcystic,” ^{1, 4–6, 8, 9, 19, 20, 22} the phenomenon may be more properly termed “microcystoid” (the suffix “oid” meaning “like”). The term “microcystoid” better characterizes the phenomenon as having characteristics similar to, but entirely different from, cysts, and reflects that the vacuolated spaces found in MMD do not have epithelial cell linings and are merely spaces between cells in the INL (Figure 3). This lack of epithelial lining is apparent at the light microscopic level. Electron microscopic evaluation confirms this and additionally shows that many very small microcysts occur that are not likely to be picked up with in vivo OCT imaging. To our knowledge this is the first report of ultrastructural evaluation of MMD in the literature.

In NHPs (and apparently in humans, as well), RNFL/RGC loss appears to be a necessary but not sufficient condition associated with MMD. Other conditions, such as epiretinal membrane traction and cystoid macular edema can produce effects that masquerade as MMD but are easily distinguished by the presence of an epiretinal membrane on OCT or ophthalmologic exam in the case of the former and foveal involvement and fluorescein leakage in the case of the latter. ²⁷

Seven of our thirteen NHPs with chronic EG did not have MMD at the time of OCT scanning. In a similar vein, in a study of human eyes with glaucoma, the presence of MMD as documented by AOSLO varied over time. ²⁸ Likewise, MMD did not manifest in every NHP examined with OCT scanning and, where it occurred, it was not observed with OCT until two or more weeks after marked RNFL thinning had been observed after HEA (Figure 6). A several week delay in apoptotic RGC death has been described following optic nerve transection in NHPs. ³⁶ This association has been described in humans, in which decreases in RNFL thickness and optic neuropathy, especially in relation to MS has been reported. ^37–40 In addition, while not found to be statistically significant in the HEA NHPs (Figure 7), the trending increase in INL thickness appears to coincide with decreasing RNFL thickness, also as previously documented in humans. ^{41, 42} RGC loss has been found to correlate with decreases in RNFL thickness in rodents, ^43–45 and to be a common etiological factor in human conditions in which MMD has been identified. ^{25, 46, 47} It has been hypothesized that trans-synaptic degeneration may be a possible cause for MMD. ^{1, 19} The observation that MMD is only observable on OCT after marked decrease in RNFL thickness (and, by extension, a slight but trending increase in INL thickness) may indicate that the trans-synaptic degeneration is retrograde in nature. A more extensive study into the relationship between RGC’s and MMD could provide more conclusive evidence of a potential causative relationship between trans-synaptic degeneration and MMD.

Despite such a potential relationship, it is worth noting that MMD in and of itself is not a pathognomonic indicator of toxicity or inflammation. Although there may be an association between the trending increase in INL thickness and the presence of MMD, histology and fluorescein angiography of these microcystoid spaces (Figures 3 and 5) show that the spaces themselves are not due to vascular leakage (e.g., from inflammation), but rather to extracellular vacuolization of the tissue itself. A strong possibility, due to the presence of smaller microcystoids shown in Figure 3, is that microcystoids first manifest at a size smaller than cells within the INL and then merge with other microcystoids until they are larger enough to be visible in in vivo OCT scanning. This vacuolization could explain the observed trend and shows that MMD is not useful as an indicator of inflammation--especially considering the hypothesis of trans-synaptic degeneration (as opposed to, for example, vascular leakage). At the same time, since the available data suggest that MMD is directly preceded by RGC loss, MMD does not appear to be a unique predictor of optic nerve injury; any condition that leads to MMD will also have already resulted in RGC loss (and, likely, RNFL thinning) by the time MMD is evident in in vivo structural or histological examinations. As such, although MMD may correlate with higher disability scores and/or lower visual acuity, ^{2, 5} it is not in and of itself a useful or unique biomarker or severity indicator.

The absence of inflammation in our NHPs is perhaps not surprising considering the work of Quigley et al showing that RGC death in experimental glaucoma and surgical axotomy occurs by apoptosis. ⁴⁸ Apoptosis is a means of programmed cell death with which organisms can remove unneeded cells by a controlled mechanism that does not trigger an inflammatory response. ^{49, 50} Although transient localized inflammation may have occurred at the site of the endodiathermy in our HEA model, we did not observe either vascular leakage on fluorescein angiography when done after several months or histologic indicators of inflammation.

MMD in NHPs share many, if not all, of the characteristics of MMD present in various human optic neuropathies. Therefore, the NHP is an appropriate animal model for further studies into the etiology of MMD in humans and may be a valuable tool for better understanding the underlying cellular mechanisms of RGC degeneration. However, the work with NHPs thus far indicates that MMD is neither predictive of RNFL/RGC loss nor an indicator of retinal inflammation, which limits its usefulness as a clinical biomarker. Even so, the human clinician should understand the frequent association of MMD and RNFL/RGC injury and the morphological characteristics that distinguish it from signs of inflammation, such as cystoid macular edema (CME). For example, CME does not spare the fovea and shows marked late leakage on fluorescein angiography. Likewise, in preclinical drug studies, the presence of MMD should not be regarded as an independent indicator of toxicity.

Data availability statement

The data that support the findings of this study are available from the corresponding author, TMN, upon reasonable request.