Ultrastructural characterization of the optic pathway in a mouse model of neurofibromatosis-1 optic glioma

Abstract

The purpose of this study was to investigate the progression of changes in retinal ganglion cells and optic nerve glia in neurofibromatosis-1 (NF1) genetically-engineered mice with optic glioma. Optic glioma tumors were generated in Nf1+/- mice lacking Nf1 expression in GFAP+ cells (astrocytes). Standard immunohistochemistry methods were employed to identify astrocytes (GFAP, S100beta), proliferating progenitor cells (sox2, nestin), microglia (Iba1), endothelial cells (CD31) and retinal ganglion cell (RGC) axons (Neurofilament 68k) in Nf1+/-, Nf1(GFAP)CKO (wild-type mice with Nf1 loss in glial cells), and Nf1+/-(GFAP)CKO (Nf1+/- mice with Nf1 loss in glial cells) mice. Ultrastructural changes in the optic chiasm and nerve were assessed by electron microscopy (EM). RGC were counted in whole retina preparations using high-resolution, mosaic confocal microscopy following their delineation by retrograde FluoroGold labeling. We found that only Nf1+/-(GFAP)CKO mice exhibited gross pre-chiasmatic optic nerve and chiasm enlargements containing aggregated GFAP+/nestin+ and S100beta+/sox2+ cells (neoplastic glia) as well as increased numbers of blood vessels and microglia. Optic gliomas in Nf1+/-(GFAP)CKO mice contained axon fiber irregularities and multilamellar bodies of degenerated myelin. EM and EM tomographic analyses showed increased glial disorganization, disoriented axonal projections, profiles of degenerating myelin and structural alterations at nodes of Ranvier. Lastly, we found reduced RGC numbers in Nf1+/-(GFAP)CKO mice, supporting a model in which the combination of optic nerve Nf1 heterozygosity and glial cell Nf1 loss results in disrupted axonal-glial relationships, subsequently culminating in the degeneration of optic nerve axons and loss of their parent RGC neurons.

Fig. 1

Neoplastic astrocytes in the pre-chiasmatic optic nerves of Nf1+/−^GFAPCKO mice. Sections from 9-month-old wild-type (A, E, I, M, Q, U), Nf1+/− (B, F, J, N, R, U), Nf1^GFAPCKO (C, G, K, O, S, U, V) and Nf1+/−^GFAPCKO (D, H, L, P, T, U, W) mice. Gross appearance of representative optic chiasm/nerves from these mice. No pathological changes were observed in wild-type (A), Nf1+/− (B) or Nf1^GFAPCKO mice (C), whereas Nf1+/−^GFAPCKO mouse optic nerves showed gross thickening and focal enlargement in the pre-chiasmatic optic nerve and chiasm (D; Asterisk). No nestin-positive astrocytes with elongated processes were observed in the prechiasmatic optic nerves and chiasm of wild-type (E, I), Nf1+/− (F, J) and Nf1^GFAPCKO mice (G, K), In contrast, the pre-chiasmatic optic nerves and chiasm of Nf1+/−^GFAPCKO mice have irregularly-shaped and thickened nestin- and GFAP-double positive cells (H, L, Arrows.). Rare sox2-positive cells with elongated processes were found in the pre-chiasmatic optic nerves and chiasm of wild-type (M, Q), Nf1+/− (N, R) and Nf1^GFAPCKO mice (O, S). In contrast, the pre-chiasmatic optic nerves and chiasm from Nf1+/−^GFAPCKO mice had increased numbers of sox2- and S100b-double positive cells (P, T, Arrowheads). Quantitation of sox2+ cells is shown in panel U. Error bar=SD. * P < 0.05 compared to wild-type mice, n = 5 optic nerve sections/mouse/group. The axons and glial cells in the pre-chiasmatic optic nerves and chiasm of Nf1^GFAPCKO mouse were indistinguishable from wild-type mice (Fig. 1V). In contrast, comparable regions from Nf1+/−^GFAPCKO mice showed hypercellular tumor cell nests and focal disruption of optic nerve fiber tracts (Fig. 1W). Scale bars: (A-D) 100 μm; (E-H, M-P) 50 μm; (I-L, Q-T) 20 μm; (V, W) 2 μm.

Fig. 2

Increased microglia in the pre-chiasmatic optic nerves of Nf1+/−^GFAPCKO mice. Sections from 9-month-old wild-type (A, E), Nf1+/− (B, F), Nf1^GFAPCKO (C, G) and Nf1+/−^GFAPCKO (D, H) mice were immunostained with Iba1 and GFAP antibodies. Ramified, quiescent microglia were observed in wild-type (A, E), Nf1+/− (B, F) and Nf1^GFAPCKO mice (C, G). In contrast, increased numbers of microglia with thickened processes were found in the pre-chiasmatic optic nerves from Nf1+/−^GFAPCKO mice (D, H). Quantitation is shown in panel I. Error bar=SD. * P < 0.05 compared to wild-type mice, n = 5 optic nerve sections/mouse/group. Scale bars: (A-D) 50 μm; (E-H) 10 μm.

Fig. 3

Increased numbers of endothelial cells in the pre-chiasmatic optic nerves of Nf1+/−^GFAPCKO mice. Sections from 9-month-old wild-type (A), Nf1+/− (B), Nf1^GFAPCKO (C) and Nf1+/−^GFAPCKO (D) mice were stained with CD31 antibodies. Few scattered small blood vessels were observed in wild-type (A), Nf1+/− (B) and Nf1^GFAPCKO (C) mouse optic nerves. In contrast, increased numbers of CD31+ cells were found in the pre-chiasmatic optic nerves of Nf1+/−^GFAPCKO mice (D, H). Quantitation is shown in panel E. Error bar=SD. * P < 0.05 compared to wild-type mice, n = 5 optic nerve sections/mouse/group. Scale bars: (A-D) 10 μm.

Fig. 4

Abnormal axon architecture in Nf1+/−^GFAPCKO mice. Sections from 9-month-old wild-type (A, E, I, M), Nf1+/− (B, F, J, N), Nf1^GFAPCKO (C, G, K, O) and Nf1+/−^GFAPCKO (D, H, L, P) mice were double labeled with Neurofilament 68k and GFAP antibodies. Well-oriented axonal fiber projections and GFAP-positive astrocytes with elongated processes were present in the pre-chiasmatic optic nerves of wild-type (A, E, M), Nf1+/− (B, F, N), and Nf1^GFAPCKO (C, G, O) mice. In contrast, comparable regions from Nf1+/−^GFAPCKO mice revealed disoriented axonal fiber projections, and axonal swelling and bulbs (D, H, P), as well as irregularly-shaped and thickened GFAP-positive cells (D, L, P). Scale bars: (A-D) 10 μm; (E-P) 5 μm.

Fig. 5

Axonal and myelin degeneration in pre-chiasmatic optic nerves of Nf1+/−^GFAPCKO mice. Cross (A, B) or longitudinal (C-I) sections from 9-month-old Nf1^GFAPCKO (A, C, E) and Nf1+/−^GFAPCKO (B, D, F, G) mice were stained with FluoroMyelin (green). The cross section of pre-chiasmatic optic nerves from Nf1+/−^GFAPCKO mice exhibited the severe alterations such as disruption of the myelin, hypermyelination, spherical lamellar bodies and focal disruption of the axon bundles (B) compared to Nf1^GFAPCKO mice. EM thin sections (C, D) and 3D projections of EM tomograms (E, F) demonstrate linear optic nerve axon fiber orientation in Nf1^GFAPCKO 9 month-old mice (C, E). In contrast, comparable regions in Nf1+/−^GFAPCKO mice showed disorientated fiber projections with degeneration of axonal profiles and myelin (D, F). A representative axon is shown with well developed internodal lamellar spheroids (F). The node and paranode regions in Nf1^GFAPCKO mice were indistinguishable from wild-type mice (G), whereas the paranode organization was disrupted in Nf1+/−^GFAPCKO mouse optic nerves. Nodal blebs correlated with the loss of subaxolemmal density (H) and the accumulation of dense bodies within swollen axons (I). Scale bars = (A, B) 10 μm; (C-I) 2μm.

Fig. 6

RGC neuronal loss in Nf1+/−^GFAPCKO mice. Representative retinal flat-mounts and sections from 9-month-old wild-type (A, B), Nf1+/− (C, D), Nf1^GFAPCKO (E, F) and Nf1+/−^GFAPCKO (G, H) mice. Compared to wild-type, Nf1+/−, and Nf1^GFAPCKO mice, Nf1+/−^GFAPCKO mouse retina showed a significant decrease in the numbers of RGC neurons. Scale bars = (A, C, E, G) 100 μm; (B, D, F, H) 20μm.

Fig. 7

Quantification of RGC neuronal loss in Nf1+/−^GFAPCKO mice. Nf1+/−^GFAPCKO mice had greater RGC neuron loss compared to wild-type, Nf1+/− and Nf1^GFAPCKO mice. Error bar=SD. *p < 0.05 compared with wild-type mice, n = 4 retinal flat-mounts/mouse/group).