A genetic mouse model of adult-onset, pervasive central nervous system demyelination with robust remyelination

Abstract

Adult-onset demyelinating disorders of the central nervous system represent the most common neurological abnormalities in young adults. Nevertheless, our understanding of disease pathogenesis and recovery in demyelinating disorders remains incomplete. To facilitate investigation into these processes, we have developed a new mouse model system that allows for the induction of dipththeria toxin A subunit expression in adult oligodendrocytes, resulting in widespread oligodendrocyte loss and demyelination of the central nervous system. These mice develop severe ataxia and tremor that correlates with impaired axonal conduction in the spinal cord. Strikingly, these animals fully recover from their motor and physiological defects and display extensive oligodendrocyte replenishment and widespread remyelination. This model system demonstrates the robust reparative potential of myelin in the central nervous system and provides a promising model for the quantitative assessment of therapeutic interventions that promote remyelination.

Figure 1

Tamoxifen-induced oligodendrocyte ablation in DTA mice. (A) Tamoxifen injected into DTA mice activates Plp-driven CreER^T-mediated deletion of the loxP-flanked eGFP, PGK-Neo and tpA coding sequences from the ROSA26-eGFP-DTA allele, resulting in DT-A expression in oligodendrocytes. (B) RT–PCR using the primer pair P1 and P2 depicted in A amplified the ∼650-bp DT-A recombination product from total RNA samples isolated from the brains of DTA mice at 7 and 14 dpi, but not from samples extracted from DTA mice at 21 dpi or control ROSA26-eGFP-DTA mice. A region of cyclophilin was amplified from all samples as an internal control. (C) Quantification of CC-1-immunostained cells at 7, 14 and 21 dpi showed that significant oligodendrocyte loss had occurred in all regions of the DTA CNS by 21 dpi (n = 4, *P < 0.04, **P < 0.003). (D) Quantitative RT–PCR analysis demonstrated that both Mbp and Plp mRNA levels were severely reduced in the brains of DTA mice at 7, 14 and 21 dpi (n = 3–5, **P < 0.003). (E) DTA mice showed higher numbers of BrdU-positive cells (green) in the cervical cord at 7 dpi as compared with control mice. Both genotypes were injected with BrdU at 5 dpi. (F) The BrdU (green, arrows) was detected in oligodendrocytes positively labelled for CC-1 expression (red, arrows), as well as in adult oligodendrocyte progenitors positively labelled for PDGFRα expression (red, arrows). Graphs in C and D indicate mean ± SD. Scale bars: 100 µm (E), 20 µm (F). Abbreviations: BS = brainstem; CB = cerebellum; CC = corpus callosum; CX = cortex; CSC-WM = cervical cord ventral white matter; CSC-GM = cervical cord grey matter.

Figure 2

Recovery of oligodendrocyte numbers and myelin gene-expression levels in DTA mice at 70 dpi. (A) Quantification of CC-1 positive cells in the brainstem, cerebellum, cervical cord grey matter and optic nerve showed that oligodendrocyte numbers were significantly reduced in DTA mice at 21 dpi, then increased slightly in all areas at 35 dpi and reached values comparable to controls everywhere at 70 dpi. (n = 4, *P < 0.013, **P < 0.006, two-tailed t-test). (B) Changes in Mbp and Plp mRNA expression levels in the brain of DTA mice over time correlated with the variation in oligodendrocyte numbers (n = 3–4, *P < 0.047, **P < 0.001, two-tailed t-test). (C) Counts of PDGFRα-stained cells in the brainstem and cerebellum of DTA mice at 21, 35 and 70 dpi demonstrated that adult OPC numbers were significantly increased at 35 dpi as compared with controls (n = 3–4, *P < 0.04, two-tailed t-test). Graphs in A–C indicate mean ± SD.

Figure 3

Progression of the myelin defects in the CNS of DTA mice. (A) Toluidine blue-stained sections showed vacuoles (arrows) within the white matter areas of the DTA CNS examined at 21 dpi. Vacuolation becomes much more severe in all areas at 35 dpi, and is ameliorated at 70 dpi. (B) Electron microscopy analysis of the DTA cervical cord white matter revealed that vacuoles are formed by the splitting of the myelin sheath lamellae (arrow) at 21 dpi. The majority of myelinated fibres look intact. By 35 dpi, myelin loss has progressed and domains containing large numbers of demyelinated axons can be observed. In addition, the large-diameter demyelinated axons show swellings with unusually high numbers of mitochondria. In the same area, myelin repair appears to occur by 70 dpi, when many axons are found to be thinly myelinated. Scale bar: 20 µm (A, cerebellum, brainstem, spinal cord), 10 µm (A, optic nerve), 2 µm (B).

Figure 4

Microglial cell numbers are markedly increased in the CNS of DTA mice. (A) Immunostaining for the microglial and monocyte/macrophage cell marker CD11b showed that positive staining (red) was significantly increased in all DTA CNS areas examined at 35 dpi as compared with 21 and 70 dpi. Cell nuclei were counterstained with DAPI (blue). (B–E) Total CNS cells were isolated from the brains and spinal cords of individual littermate control and DTA mice (n = 8) at 35 dpi. The cells were enumerated and analysed via flow cytometric analysis, gating on live cells for the presence of CD45⁺ cells and gating on the CD45^hi (blue) versus CD45^lo (purple) populations for the presence of CD11b⁺ cells. Flow plots from a representative littermate control mouse, shown in B, and a DTA mouse, in C, are presented. The total number of CD45^hi and CD45^lo cells, shown in D, and the total number of CD45^lo/CD11b⁺ and CD45^hi/CD11b⁺ cells, shown in E of control versus DTA animals are shown. The data are presented as the average of the cell counts from eight mice per group ± SEM in D and E. Scale bar: 100 µm (A).

Figure 5

Quantification of myelin and axonal defects in the optic nerves of DTA mice. (A) Electron microscopy analysis of the optic nerve revealed that the extensive myelin loss observable in DTA mice at 56 dpi is repaired by 70 dpi, at which point many axons were found to be thinly myelinated. (B) Although the numbers of unmyelinated axons were significantly higher in DTA mice at both time points, there were ∼70% fewer unmyelinated axons at 70 dpi as compared with 56 dpi (**P < 1.30 × 10⁻⁵, n = 3). (C) Linear regression analysis comparing the g-ratios and axonal diameters of myelinated axons in the optic nerve showed thinner myelin for all sizes of axons in DTA mice at 70 dpi (black dots, n = 3) as compared with controls (grey dots, n = 3). (D) The total numbers of axons in the optic nerves of DTA mice were found to be similar to control values at 21 and 70 dpi (P > 0.27, n = 3). Graphs in B and D indicate mean ± SD. Scale bar: 2 µm (A).

Figure 6

Assessment of phenotypic progression in DTA mice. (A) SSEP waveforms. Each SSEP trace represents the average of 25–30 evoked responses recorded from the L4–L5 intervertebral space (low lumbar level) or T5–T6 intervertebral space (mid-thoracic level) following stimulation of the tibial nerve at the ankle. (B) Statistical analysis of the SSEP parameters: peak latency (Lat) and Δlatency (ΔLat, difference between the T5–T6 and L4–L5 peak latencies) and amplitude (Amp). In DTA mice at 35 dpi, peak latencies are prolonged and Δlatencies are increased, while amplitudes are decreased, indicating severe conduction defects, both in the PNS and CNS of these mice. At 70–77 dpi, the amplitudes observed in DTA mice reach values comparable to controls, but though improved in comparison to 35 dpi values, a milder defect in peak latency time lingers. At 35 dpi, three of the eight DTA mice did not demonstrate recordable T5–T6 responses, indicating a central conduction failure. The statistical analysis of T5–T6 recordings does not include those with absent responses. Graphs indicate mean ± SEM. For 35 dpi, n = 7–8, *P < 0.005, **P < 0.0001. For 70–77 dpi, n = 6–7, *P < 0.03; **P < 0.008. (C) The DTA mice were unable to perform on the rotarod at 35 and 42 dpi. They began showing a gradual improvement at 49 dpi that continued until 77 dpi, when their latencies became similar to control mice. Graphs indicate mean ± SD, n = 5 mice for DTA and n = 4 for controls, *P < 0.08, **P < 0.007 (two-tailed t-test).