Spontaneous optic nerve compression in the osteopetrotic (op/op) mouse: a novel model of myelination failure

Abstract

We report a focal disturbance in myelination of the optic nerve in the osteopetrotic (op/op) mouse, which results from a spontaneous compression of the nerve resulting from stenosis of the optic canal. The growth of the op/op optic nerve was significantly affected, being maximally suppressed at postnatal day 30 (P30; 33% of age matched control). Myelination of the nerve in the optic canal was significantly delayed at P15, and myelin was almost completely absent at P30. The size of nerves and myelination were conserved both in the intracranial and intraorbital segments at P30, suggesting that the axons in the compressed site are spared in all animals at P30. Interestingly, we observed recovery both in the nerve size and the density of myelinated axons at 7 months in almost half of the optic nerves examined, although some nerves lost axons and became atrophic. In vivo and ex vivo electrophysiological examinations of P30 op/op mice showed that nerve conduction was significantly delayed but not blocked with partial recovery in some mice by 7 months. Transcardial perfusion of FITC-labeled albumin suggested that local ischemia was at least in part the cause of this myelination failure. These results suggest that the primary abnormality is dysmyelination of the optic nerve in early development. This noninvasive model system will be a valuable tool to study the effects of nerve compression on the function and survival of oligodendrocyte progenitor cells/oligodendrocytes and axons and to explore the mechanism of redistribution of oligodendrocyte progenitor cells with compensatory myelination.

Figure 1.

The optic nerve of op/op mice is compressed at the optic canal. A, The optic nerve of 7-day-old op/op mouse. The nerve is translucent, indicating that myelination has not begun or is incomplete and compression of the nerve is not seen at the optic canal (arrowhead). B, Optic nerves on the cranial base of the op/op mouse at P30. The nerve compression coincided with the optic canal (arrows). The left compressed nerve was exposed by removing the sphenoid bone, showing that the compression was confined to the intracanalicular segment. OC, Optic chiasm; IC, intracranial nerve; IO, intraorbital nerve. C, Bilateral compression of the optic nerve of op/op mouse at P30, removed from the cranium. The compressed areas were translucent, indicating lack of myelin. D, Chronologically, aligned optic nerves showed that the compression in op/op mice was present from P10 to P60, whereas the noncompressed wild-type (wt) nerve is not compressed and is normally myelinated at the optic canal. Scale bar: D, 1 mm.

Figure 2.

Myelination is inhibited in the intracanalicular optic nerve of op/op mice. Toluidine blue-stained cross sections from the optic nerves of op/op mice at 7, 10, 15, 30, and 60 days, and 7 months of age. Whereas myelin develops with time both in the intraorbital (left panels) and intracranial (right panels) segments, the compressed intracanalicular nerve segment (middle panels) had only sporadic myelination of axons at P15 and P30, suggesting a failure in myelination. The appearances in the intraorbital and intracranial segments were comparable with those of wild-type control (data not shown). Note that only a few cell nuclei were identified in the intracanalicular nerve at P30. Scale bars: 20 μm; insets, 200 μm.

Figure 3.

Nerve area, axon myelination, and glial cell number are reduced in the compressed portion of the op/op optic nerve. A, Chronological changes in the cross-sectional area of op/op optic nerves. The areas of optic nerve cross section increase with time in areas of wild-type mice and the intraorbital and intracranial segments of op/op mice. However, the growth of optic nerve was suppressed in the intracanalicular segment of op/op mice from 7 through 60 d and showed partial recovery at 7 months. Data are mean ± SEM of 6 nerves from 3 animals. B, Chronological changes in the density of myelinated fibers in op/op optic nerves. The lack of myelin was most pronounced at P30 in the intracanalicular segment of op/op optic nerve, and myelination slowly proceeds thereafter. Data are mean ± SEM of 6 nerves from 3 animals. Significance was measured by one-way ANOVA followed by Scheffé's F post hoc test. Chronological changes in the number of glial cells in the intraorbital (C), intracanalicular (D), and intracranial (E) segments of the optic nerve. A significant decrease in glial number was seen only in the intracanalicular segment of op/op mice at P10, P15, P30, and P60, reaching the maximum reduction at P30. *p < 0.05, wild-type and op/op mice at the same age (one-way ANOVA followed by Bonferroni's post hoc test). **p < 0.01, wild-type and op/op mice at the same age (one-way ANOVA followed by Bonferroni's post hoc test). ***p < 0.001, wild-type and op/op mice at the same age (one-way ANOVA followed by Bonferroni's post hoc test).

Figure 4.

The compressed nerve of the op/op mouse lacks glia at P60, although axons are unaffected. A, At P60 the intracanalicular segment of the optic nerve lacks glial cell nuclei in a majority of the nerve. A portion of the nerve in which glial cells are present coincides with a small number of adjacent myelinated fibers (*; enlarged in B). C, By contrast, the intracranial portion of the same nerve has a normal density of myelinated fibers, confirming the presence of a normal axon density along the entire nerve (*; enlarged in D). Scale bars: A, C, 50 μm; B, D, 5 μm.

Figure 5.

Electron micrographs of the compressed optic nerve of op/op mice at P30. Most axons were myelinated in the intraorbital (A) and intracranial (data not shown) segments. However, the majority of axons were unmyelinated in the intracanalicular segment, and in this nerve only one myelinated axon (arrow) is present (B). Myelin debris or degenerating axons were not seen. Scale bars: A, 2 μm; B, 1 μm.

Figure 6.

At 7 months, the optic nerves in op mice show variable recovery. In the first mouse (A, D, H), the right optic nerve cannot be seen in situ (A), yet when trimmed and embedded it can be seen that the nerve is atrophic (D) and degenerates with only scattered myelinated axons present (H). In the second mouse (B, E, F, I, J), the left nerve has recovery of nerve area and axon myelination, whereas the right nerve is noticeably smaller (B, F), though well myelinated (J), possibly through the loss of some axons. In the third mouse (C, G, K), both the left and right optic nerves remain compressed at the canal, and there were only scattered myelinated fibers, yet axons appear to remain intact (G, K). Scale bars: D–G, 100 μm; H–K, 10 μm.

Figure 7.

Glial cells are reduced in the compressed optic nerve of op/op mice at P30, but axons are preserved. Immunohistochemistry on the longitudinal sections (A–D, G, H) and cross sections (E, F) of optic nerves from wild-type and op/op mice at P30 (E, F are from op/op mice only). A, MBP immunolabeling showed the absence of myelin in the compressed lesion of op/op mice. B, Neurofilament (200 kDa) is positive in the entire length of optic nerves and highly immunoreactive in the compressed lesion because the nerve fibers converge in the area and probably because the lack of myelin favored reaction of the antibody. Mature oligodendrocytes (C, E) and OPCs (D, F) were immunolabeled by anti-GST-π antibody and anti-NG2 antibody, respectively. GST-π revealed diffuse staining of myelin as well as strong immunoreactivity in the oligodendrocyte cell bodies, both of which were absent in the intracanalicular segment of the op/op optic nerve (C–F). GFAP immunolabeling shows relative absence of processes and cell bodies of astrocytes in the core lesion of the compressed optic nerve. G, In the periphery, however, GFAP immunoreactivity is dense with abundant astrocytic processes. H, The number of microglia and macrophages was highly reduced in the op/op optic nerve, although the compressed lesion contained some activated microglia (or macrophages).

Figure 8.

TUNEL and Ki67 antigen detection did not reveal the mechanism of cell reduction in the intracanalicular optic nerve of op/op mice, but the compressed optic nerve of op/op mice is potentially ischemic. A, The number of TUNEL-positive apoptotic cells in the optic nerve of op/op mice was higher than that of wild-type mice in all regions and at all time points examined. B, There was no difference in the number of Ki67-positive proliferating cells between wild-type and op/op mice. Data are presented as an index (number of TUNEL- or Ki67-positive cells divided by integrated DAPI signals in each region). n = 6 nerves from 3 animals per group. C, The optic nerve blood flow was estimated as the total FITC-positive pixel areas in 50 μm cross sections of the optic nerve of 30-day-old wild-type and op/op mice transcardially perfused with FITC-conjugated BSA in gelatin. D, Percentage vascular area is shown by dividing the FITC-positive pixels (as in C) over the pixel area of the cross section. Both graphs indicated significant reductions of the estimated blood flow in op/op mice at the intracanalicular segment and 0.5 mm distal to the site. Data are expressed as mean ± SEM (n = 6 nerves per group) at every 0.5 mm from the intracanalicular segment (canal) toward the intraorbital segment (positive values) and the intracranial segment (negative values). *p < 0.05, compared with wild-type control in the same region (one-way ANOVA followed by Bonferroni's post hoc test). **p < 0.01, compared with wild-type control in the same region (one-way ANOVA followed by Bonferroni's post hoc test).

Figure 9.

Compressed optic nerve of op/op mice is functionally impaired in vivo. A–H, Representative ERG and VEP traces recorded simultaneously in response to a medium intensity flash for a wild-type (A–D) and an op/op mouse (E–H). At 30 d, the b-wave of the photopic ERG (arrow) is robust in both the control (A) and op/op mouse at 30 d (E). At 7 months, ERGs remained stable in the control (C) and op/op mouse (G). In the control, the N1 wave of the flash VEP is very low amplitude but clearly present at 30 d (B) and increased markedly in amplitude with development (arrow) (D). In contrast, the flash VEPs in the op/op mouse were very low amplitude at 30 d (F), yet a VEP was clearly present at 7 months (H), albeit with reduced amplitude and increase peak times. Calibration: 100 mV, 50 ms.

Figure 10.

Compressed optic nerve of op/op mice is functionally impaired in vitro. A, Compound action potential of wild-type (n = 4 nerves, P30; n = 4 nerves, 7 months) and op/op (n = 4 nerves, P30; n = 4 nerves, 7 months) mice, measured with suction electrodes and brief bipolar stimulation at P30 and 7 months. Notice a longer time to peak from the stimulation artifact for op/op at P30 but not 7 months of age, reflecting the reduced conduction velocity at P30 compared with the wild-type. Also note a smaller amplitude in the P30 op/op mice, reflecting that many axons are not conducting action potentials (room temperature 21°C). B, Velocity of compound action potentials in wild-type and op/op mice at P30 and 7 months of age. At P30, the wild-type velocity was 1.7 ± 0.16 m/s (n = 4) and op/op velocity was slower, at 0.75 ± 0.2 m/s (n = 4). C, Refractory period analysis of compound action potentials at P30 and 7 months. Refractory period measures how fast a second action potential recovers from a first stimulation. This recovery of the second action potential was assayed using standard twin-pulse stimulations with increasing interpulse intervals. Note that at P30 the curve for op/op mice (open symbol) was right shifted to the curve for wild-type mice (closed symbols). The average 50% recovery was 24 and 29 s for wild-type and op/op mice, respectively. The slight increase in refractory period (from 24 to 29 s) for op/op mice was consistent with a slower conduction velocity (B). D, Axonal calcium response at P30 evoked by repetitive action potentials (2 s at 80 Hz). The Ca response was expressed as changes relative to the resting level (ΔF/F). Response of the P30 op/op nerve was measured at two locations for each nerve: at the unconstricted and the constricted region. Within the same op/op nerve, the Ca response was smaller at the constricted region (ΔF/F = 0.017 ± 0.002, n = 4) compared with the unconstricted region (ΔF/F = 0.038 ± 0.005, n = 4). The wild-type response was ΔF/F = 0.062 ± 0.006 (n = 4). Because the Ca response was measured from a large population of axons, the smaller response at the constricted region suggests either conduction block or reduced conduction at the constriction site. E, Actual axonal Ca response traces in the wild-type and op/op nerves. In the same op/op nerve, the amplitude of the Ca response is smaller at the constricted site compared with the unconstricted site. Further, it is interesting to note that the poststimulation Ca decline is slower at the constricted site (see inset; Ca responses normalized to the same amplitude). Because poststimulation Ca decline is determined by Ca buffering (Ca extrusion and/or Ca sequestration by intracellular organelles), the slower Ca decline at the constriction site (inset) suggests a selective abnormality in local Ca buffering at the constriction site of the op/op nerve. **p < 0.01, compared with wild-type control. ***p < 0.001, compared with wild-type control.

Figure 11.

Lack of model apparatus in the op/op mouse at the canal. Representative images of double immunofluorescent staining for voltage-gated sodium ion channel Nav1.6 (green) and contactin-associated protein (Caspr; red) (A) in the intracranial (B) and intracanalicular (C) segments of optic nerve in op/op mice at P30. Note that the normal nodal expression of Nav1.6 and paranodal expression of Caspr was lost in the intracanalicular segment, whereas they were maintained in other regions, suggesting that normal saltatory conduction was disturbed in the intracanalicular segment. Scale bars: A, 500 μm; B, C, 10 μm.