Normal tension glaucoma-like degeneration of the visual system in aged marmosets

Abstract

The common marmoset (Callithrix jacchus) is a non-human primate that provides valuable models for neuroscience and aging research due to its anatomical similarities to humans and relatively short lifespan. This study was carried out to examine whether aged marmosets develop glaucoma, as seen in humans. We found that 11% of the aged marmosets presented with glaucoma-like characteristics; this incident rate is very similar to that in humans. Magnetic resonance imaging showed a significant volume loss in the visual cortex, and histological analyses confirmed the degeneration of the lateral geniculate nuclei and visual cortex in the affected marmosets. These marmosets did not have elevated intraocular pressure, but showed an increased oxidative stress level, low cerebrospinal fluid (CSF) pressure, and low brain-derived neurotrophic factor (BDNF) and TrkB expression in the retina, optic nerve head and CSF. Our findings suggest that marmosets have potential to provide useful information for the research of eye and the visual system.

Figure 1

Evaluation of ocular fundus images of marmosets. (a) Ocular fundus photographs of aged control marmoset (No. 30 in Supplementary Table 1). (b–e) Ocular fundus photographs of glaucomatous marmosets. No. 33 (b), No. 34 (c), No. 35 (d), and No. 36 (e). Optic disc cuppings are outlined by dashed lines and the cup/disc ratio are shown in parentheses. The edge of the cupping was traced from the 3D images of the optic nerve head obtained by OCT and the lines were superimposed on the fundus photograph.

Figure 2

Degeneration of the optic nerve and retina, and impaired retinal function in glaucomatous marmosets. (a) In vivo imaging of the optic disc by vertical scan through the centre of the optic disc by SD-OCT. Arrowheads indicate the cupping of the optic disc and dotted lines indicate the LC. LC: lamina cribrosa, GCC: ganglion cell complex. (b,c) Quantitative analysis of the thickness of the LC (b) and GCC (c). n = 28 (aged) and 6 (glaucoma). (d) Three-dimensional plots of the retinal responses as examined by multifocal electroretinogram (mfERG). A higher score (white) indicates highly sensitive visual function. Values are given in nV per square degree (nV/deg²). (e) Quantitative analysis of the visual responses. n = 28 (aged) and 6 (glaucoma). (f) H&E staining of the optic nerve head. Enhanced optic disc cupping (arrowheads) and thinning of the LC (dotted lines) are apparent in the glaucomatous marmoset. Scale bar: 200 µm. (g) Quantitative analysis of the LC thickness. n = 3 per group. (h) H&E staining of the retina. Inner retinal degeneration is observed in the glaucomatous marmoset. The mid-peripheral region is shown. Scale bar: 100 µm. GCL: ganglion cell layer. (i,j) Quantitative analysis of the cell number in the GCL (i) and GCC thickness (j). n = 3 per group. The data are presented as means ± S.E.M. *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 3

Pathological features resembling human normal tension glaucoma in aged marmosets. (a,b) In vivo imaging of the anterior chamber by SD-OCT. Illustration of the parameters measured in the anterior chamber (a). The iridocorneal angle in the glaucomatous marmoset is wide open and comparable to that in aged marmoset (b). ACA: anterior chamber angle, AOD: angle opening distance. (c,d) Quantitative analyses of ACA (c) and AOD750 (d) in aged and glaucomatous marmosets. n = 28 (aged) and 6 (glaucoma). (e) Quantitative analysis of the axial length in aged and glaucomatous marmosets using a color Doppler imaging scanner. n = 30 (aged) and 6 (glaucoma). (f) H&E staining of aqueous humor drainage structures in aged (left) and glaucomatous (right) marmosets. The iridocorneal angle in the glaucomatous marmoset is normal with an obvious Schlemm’s canal (arrow) and trabecular meshwork. Scale bar: 200 µm. (g) The IOP of aged and glaucomatous marmosets. IOP: intraocular pressure. n = 63 (aged) and 6 (glaucoma). (h) The CSF pressure of aged and glaucomatous marmosets. CSF: cerebrospinal fluid. n = 6 (aged) and 3 (glaucoma). (i) Representative images of the flow velocity of the ophthalmic artery using a color Doppler imaging scanner. PSV: peak systolic velocity, EDV: end diastolic velocity. (j) Quantitative analysis of the resistive index. The resistive index was calculated according to the following formula: Resistive index = 1 – (EDV / PSV). n = 24 (aged) and 5 (glaucoma). The data are presented as means ± S.E.M. *P < 0.05; **P < 0.01.

Figure 4

Genomic analysis of glaucomatous marmosets for glaucoma associated genes. (a) A single base substitution in the MYOC gene was identified in the glaucomatous marmoset (No. 34 in Supplementary Table 1). However, this is a silent mutation: a single base mutation that does not alter protein production. (b) A domain structure of the human MYOC gene with indication of the mutations associated with glaucoma. All the reported human mutation sites of the MYOC gene were analysed in the glaucomatous marmosets (No. 33–36). Gene mutations associated with glaucoma were not detected. SP: signal peptide, HtH: helix-turn-helix, CC: coiled coil, GD: globular domain, OD: olfactmedin domain. (c,d) Domain structures of human OPTN (c) and WDR36 (d) genes and location of identified mutations. All the reported human mutation sites of the OPTN gene and exons 8, 11, 13, and 17 of the WDR36 gene were analysed in the glaucomatous marmosets (No. 33–36). Gene mutations associated with glaucoma were not detected. CC: coiled coil, LZ: leucine zipper, LIR: LC3-interacting region, UBD: ubiquitin-binding domain, ZF: zinc finger, WD: WD repeat domain.

Figure 5

Follow-up studies of a glaucomatous marmoset over 12 months. (a) Ocular fundus photographs of initial examination (Year 0) and one year later (Year 1) in the glaucomatous marmoset (No. 34 in Supplementary Table 1). Dotted lines indicate the cupping of the optic disc. (b) Quantitative analysis of the vertical cup/disc ratio. R: right eye, L: left eye, Y0: year 0, Y1: year 1. (c) In vivo imaging of the optic disc by the horizontal and vertical scan through the centre of the optic disc by SD-OCT. Arrowheads indicate the cupping of the optic disc and dotted lines indicate the LC. (d) Longitudinal evaluations of the GCC thickness by a circular scan around the optic disc by SD-OCT. (e) Quantitative analysis of the LC thickness by cross-sectional images of the optic disc by SD-OCT. (f) Three-dimensional plots of the retinal responses as examined by mfERG. Values are given in nV per square degree (nV/deg²). (g) Quantitative analysis of the visual responses in (f).

Figure 6

Degeneration of the central visual system in glaucomatous marmosets. (a) Volume loss in the primary visual cortex, also known as visual area 1 (V1) in the glaucomatous marmosets comparison with normal aged controls. The color scale bar in voxel-wise statistics indicates the t-score and the areas of the gray matter with significant volume reduction were superimposed on the template brain (P < 0.001, Student’s t test). n = 4 (aged) and 3 (glaucoma). Scale bar: 10 mm. (b) Representative images of coronal sections of the lateral geniculate nuclei (LGN) (upper) and magnified images of the magnocellular (M)- and parvocellular (P)-layers of the LGN (area No. 7) in aged and glaucomatous marmosets (lower). Scale bar: 1 mm and 100 µm for upper and lower panels, respectively. (c) Quantitative analyses of the cell number in the LGN. n = 3 per group. (d) Representative images of coronal sections of the visual cortex (upper) and magnified images of the layer 4 of the visual cortex in aged and glaucomatous marmosets. Scale bar: 1 mm and 50 µm for upper and lower panels, respectively. (e) Quantitative analyses of the cell number in the layer 4 of the visual cortex. n = 3 per group. The data are presented as means ± S.E.M. ***P < 0.001.

Figure 7

Increased oxidative stress and decreased BDNF/TrkB expression in glaucomatous marmosets. (a) 4-HNE expression in the retina detected by immunohistochemistry. Scale bar: 100 µm. (b) Quantitative analyses of the intensity of 4-HNE. n = 3 per group. (c) 4-HNE expression in the blood detected by immunoblot analyses. n = 9 (aged) and 3 (glaucoma). (d) GSH concentrations in the blood. n = 4 (aged) and 3 (glaucoma). (e) BDNF and TrkB in the optic nerve head (ONH) and retina detected by immunohistochemistry. Scale bar: 100 µm. (f,g) Quantitative analyses of the intensity of BDNF (f) and TrkB (g). n = 3 per group. (h) BDNF concentrations in the CSF. n = 3 per group. The data are presented as means ± S.E.M. *P < 0.05; **P < 0.01, ***P < 0.001.