Endogenous Nkx2.2+/Olig2+ oligodendrocyte precursor cells fail to remyelinate the demyelinated adult rat spinal cord in the absence of astrocytes

Abstract

Chronic demyelination is a pathophysiologic component of compressive spinal cord injury (SCI) and a characteristic finding in demyelinating diseases including multiple sclerosis (MS). A better characterization of endogenous cells responsible for successful remyelination is essential for designing therapeutic strategies aimed at restoring functional myelin. The present study examined the spatiotemporal response of endogenous oligodendrocyte precursor cells (OPCs) following ethidium bromide (EB)-induced demyelination of the adult rat spinal cord. Beginning at 2 days post-EB injection (dpi), a robust mobilization of highly proliferative NG2(+) cells within the lesion was observed, none of which expressed the oligodendrocyte lineage-associated transcription factor Nkx2.2. At 7 dpi, a significant up-regulation of Nkx2.2 by OPCs within the lesion was observed, 90% of which coexpressed NG2 and virtually all of which coexpressed the bHLH transcription factor Olig2. Despite successful recruitment of Nkx2.2(+)/Olig2(+) OPCs within the lesion, demyelinated axons were not remyelinated by these OPCs in regions lacking astrocytes. Rather, Schwann cell remyelination predominated throughout the central core of the lesion, particularly around blood vessels. Oligodendrocyte remyelination was observed in the astrogliotic perimeter, suggesting a necessary role for astrocytes in oligodendrocyte maturation. In addition, reexpression of the radial glial antigen, RC-1, by reactive astrocytes and ependymal cells was observed following injury. However, these cells did not express the neural stem cell (NSC)-associated transcription factors Sox1 or Sox2, suggesting that the endogenous response is primarily mediated by glial progenitors. In vivo electrophysiology demonstrated a limited and unsustained functional recovery concurrent with endogenous remyelination following EB-induced lesions.

Fig. 1. EB-induced demyelination in the VLF

(A) Stereotactic injection of EB into the VLF consistently resulted in well-defined lesions as schematically illustrated. (B) LFB staining reveals a loss of myelin throughout most of the lesion after 28 days. EB is toxic to both GFAP⁺ astrocytes (C) and APC⁺ oligodendrocytes (D). Ultrastructural analysis revealed that after 7 days, many viable demyelinated axons survive EB treatment in both the peripheral (E) and central (F) portions of the lesion. Scale bar = 100 µm in (B–D); 2 µm in (E and F).

Fig. 2. NG2⁺ polydendrocyte response to EB injection

(A) NG2⁺ cells are evenly spaced in the uninjured VLF and have irregularly shaped cell bodies. (B) At 2 dpi, NG2⁺ polydendrocytes demonstrate increased NG2⁺ staining and short processes. In (B, C, and E), the larger inset box shows the entire lesioned VLF while the inner box shows the region shown at higher magnification. (C) By 7 dpi, NG2⁺ polydendrocytes exhibited complex morphologies with many branching processes. (D) NG2 did not label activated macrophages at 7 dpi as evidenced by a lack of NG2 and ED-1 coexpression. (E) Increased NG2⁺ immunoreactivity is observed in EB lesions up to 28 dpi. (F) In animals sacrificed at 2 dpi, 85% of the polydendrocytes within the lesion express BrdU following a 24-h BrdU pulse on Day 2, suggesting that the increase in polydendrocyte density within EB lesions is at least partially due to local proliferation. (G) Polydendrocyte density within EB lesions is significantly increased at 2, 7, and 28 dpi compared to uninjured controls. Data are expressed as the mean ± SD (uninjured; n = 10, Day 2; n = 9, Day 7; n = 8, Day 28; n = 8). One-way ANOVA (F = 50.5, df = 3.31, P < 0.001). Tukey post hoc (all comparisons, *P < 0.001). Scale bar = 5 µm in (A, D, and F); 8 µm in (B); 3 µm in (C and E).

Fig. 3. Nkx2.2 and Olig2 expression within EB lesions

(A) In the uninjured VLF, faint Nkx2.2 expression is observed in many cells, approximately one third of which are NG2⁺ (arrow). Many Nkx2.2⁺ nuclei are associated with NG2 processes; however, they do not meet criteria for NG2 coexpression (arrowheads). The inset shows the entire region of the VLF and the inner box shows the area presented at higher magnification. (B) At 2 dpi, Nkx2.2 is not expressed within the lesion despite the increased density of NG2⁺ polydendrocytes. (C) At 7 dpi, 90% of Nkx2.2⁺ nuclei coexpress NG2. (D–F) All Nkx2.2⁺ nuclei coexpress Olig2 at 7 dpi. The proportion of Nkx2.2⁺ cells coexpressing NG2 is significantly increased in Day 7 lesions compared to uninjured controls (G), whereas the proportion of NG2⁺ cells coexpressing Nkx2.2 is not (H). Data are expressed as the mean ± SD. *P < 0.05, t = 3.1, df = 8.4. Scale bar = 6 µm in (A); 15 µm in (B and D– F); 24 µm in (C).

Fig. 4. Nestin up-regulation by reactive astrocytes

(A) Beginning at 7 dpi, nestin is up-regulated by cells surrounding the lesion. In (A–F), the insets show the entire region of the VLF and the inner box designates the area shown at higher magnification. (B and C) GFAP double labeling reveals that nestin⁺ cells are GFAP⁺-reactive astrocytes. (D–F) Nestin up-regulation by reactive astrocytes around the lesion is maintained at 14 dpi. Nestin up-regulation by ependymal and/or subependymal cells was not observed at 7 (G) or 14 (H) dpi. Asterisks (*) identify the central canal. Scale bar = 30 µm.

Fig. 5. RC-1 up-regulation by reactive astrocytes and ependymal cells

(A) RC-1, a marker for radial glia, is up-regulated by cells surrounding EB lesions. In (A–F), the inset shows the entire region of the VLF and the inner box illustrates the areas shown at higher magnification. (B and C) GFAP double labeling reveals that RC-1⁺ cells are GFAP⁺-reactive astrocytes. (D– F) RC-1 up-regulation by reactive astrocytes around the lesion is maintained at 28 dpi. (G) RC-1 is also up-regulated by ependymal cells surrounding the central canal at 7 dpi. (H) GFAP is expressed by subependymal astrocytes, but not ependymal cells. (I) Twenty-four-hour BrdU pulse labeling at 7 dpi reveals that many ependymal cells are dividing at this time. (J) Merged image of (G– I). (K) At 28 dpi, ependymal cells no longer express RC-1. (L) GFAP expression by subependymal astrocytes at 28 dpi. (M) Twenty-four-hour BrdU pulse labeling at 28 dpi shows that ependymal cells are no longer dividing at this time. (N) Merged image (K– M). Scale bar = 30 µm in (A– F); 25 µm in (G–N).

Fig. 6. Remyelinating oligodendrocytes are restricted to the perimeter of the lesion

(A) APC⁺ oligodendrocytes are present in the perimeter of the lesion after 28 days, but not in the central region. Twenty-four-hour pulse labeling with BrdU at 2 dpi in animals, which were sacrificed at 28 dpi, reveals that (A–C) many APC⁺ oligodendrocytes in the perimeter of the lesion are BrdU⁺. (D) Triple label immunohistochemistry reveals that new oligodendrocytes are closely associated with the band of reactive astrocytes in the perimeter of the lesion. (E) Ultrastructural analysis reveals that oligodendrocytes in the perimeter of the lesion produce thin myelin, characteristic for CNS-type remyelination; these may be distinguished from the thicker, spared myelin. Scale bar = 25 µm in (A– C); 28 µm in (D); and 6 µm in (E).

Fig. 7. Schwann cell remyelination in the lesion core

(A) P₀ immunohistochemistry demonstrates considerable Schwann cell-mediated remyelination as early as 28 dpi. Schwann cell remyelination begins in a distinct perivascular pattern. (B) After 74 days, Schwann cell (S) remyelination is extensive in the central portion of the lesion while oligodendrocyte (O) remyelination is limited to the perimeter of the lesion, adjacent to normal (N), uninjured white matter. In toluidine blue-stained plastic sections, Schwann cell myelin appears darker than CNS myelin. (C) Ultrastructural analysis confirmed the presence of characteristic Schwann cell remyelination in the central core of the VLF at 28 dpi; bv = blood vessel. (D) Higher magnification of remyelinating Schwann cells in the center of Day 28 lesions. Schwann cell nuclei are indicated by arrows. Scale bar = 90 µm in (A); 30 µm in (B); 3 µm in (C); and 1 µm in (D).

Fig. 8. Endogenous remyelination fails to restore sustained electrophysiologic function

(A) A representative sample of tcMMEP responses from an animal demonstrating transient electrophysiologic functional recovery. (B) By 14 dpi, 100% of tcMMEP signals were abolished (n = 22). Electrophysiologic function returned in some lesions as evidenced by a return of tcMMEP signals. (C) Recovered tcMMEP signals demonstrated significantly increased latencies (P < 0.001, t = 10.7, df = 6) and significantly decreased amplitudes (P < 0.01, t = 4.1, df = 6) compared to baseline controls.