Neurotrophin-3 and brain-derived neurotrophic factor induce oligodendrocyte proliferation and myelination of regenerating axons in the contused adult rat spinal cord

Abstract

Functional loss after spinal cord injury (SCI) is caused, in part, by demyelination of axons surviving the trauma. Neurotrophins have been shown to induce oligodendrogliagenesis in vitro, but stimulation of oligodendrocyte proliferation and myelination by these factors in vivo has not been examined. We sought to determine whether neurotrophins can induce the formation of new oligodendrocytes and myelination of regenerating axons after SCI in adult rats. In this study, fibroblasts producing neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, nerve growth factor, basic fibroblast growth factor, or beta-galactosidase (control grafts) were transplanted subacutely into the contused adult rat spinal cord. At 10 weeks after injury, all transplants contained axons. NT-3 and BDNF grafts, however, contained significantly more axons than control or other growth factor-producing grafts. In addition, significantly more myelin basic protein-positive profiles were detected in NT-3 and BDNF transplants, suggesting enhanced myelination of ingrowing axons within these neurotrophin-producing grafts. To determine whether augmented myelinogenesis was associated with increased proliferation of oligodendrocyte lineage cells, bromodeoxyuridine (BrdU) was used to label dividing cells. NT-3 and BDNF grafts contained significantly more BrdU-positive oligodendrocytes than controls. The association of these new oligodendrocytes with ingrowing myelinated axons suggests that NT-3- and BDNF-induced myelinogenesis resulted, at least in part, from expansion of oligodendrocyte lineage cells, most likely the endogenous oligodendrocyte progenitors. These findings may have significant implications for chronic demyelinating diseases or CNS injuries.

Fig. 1.

Illustration of proportional area measurements. Images are from a cross section of an NT-3 graft that was immunohistochemically labeled for MBP. The left image shows the digitized spinal cord; the graft border has been manually outlined in red. The area inside the red border, i.e., the cross-sectional graft area, corresponds to the scan area. The right image demonstrates the MBP-ir profiles within the graft that were selected as positive (green); these correspond to the target area. Proportional area was calculated by dividing the target area by the scan area.

Fig. 2.

Neurofilament-ir in a normal, 2 d post-injury (PI) and 10 week PI spinal cord. Comparison of neurofilament-ir at T₈ from a normal spinal cord (A, 5×) and a spinal cord epicenter at 2 Days PI (B, 5×) reveals the minimal amount of axons spared in the epicenter at the time of transplantation. By 10 Weeks PI (C, 5×), there was an increase in the number of axons observed in the epicenter compared with that seen at 2 Days PI, suggesting that a limited amount of endogenous regrowth occurred at the injury site. Theblack boxes in A–C are shown at higher power (40×) to the right of each image.

Fig. 3.

Comparison of neurofilaments in injury control and transplanted spinal cords. Horizontal sections from an injured spinal cord (nongraft recipient) (A), a β-gal graft (B), and an NT-3-producing graft (C) immunolabeled for neurofilament and counterstained with cresyl violet. Images are from rats that received grafts at 10 d after injury and survived for 12 weeks. They are representative, however, of spinal cords from 2 d post-injury transplants and 10 week survival times. Arrows inB and C delineate graft perimeters. Note that grafts filled the lesion cavities and that NT-3 production increased the amount of axons extending into the grafts. Quantitation of the proportional area of the grafts occupied by neurofilaments (D) revealed that NT-3, BDNF, CNTF, and β-gal grafts contained significantly more axons than the lesion epicenter and nongraft recipients (^†p < 0.05 for β-gal and CNTF, and p < 0.001 for NT-3 and BDNF). In addition, BDNF and NT-3 stimulated a more robust infiltration of axons in comparison with β-gal or other growth factor grafts (*p < 0.01). (Bars represent mean ± SEM;n = 5 for injury control, NGF, CNTF, FGF;n = 6 for β-gal, NT-3; n = 11 for BDNF).

Fig. 4.

Immunocytochemical identification of several fiber phenotypes within the grafts. CGRP, Horizontal sections of an uninjured spinal cord (A, 20×), an injured nongrafted spinal cord (B, 20×), and a BDNF-producing graft (C, 20×). In uninjured controls, CGRP-ir was restricted to the soma and axons of ventral horn motor neurons and small diameter axons in the dorsal white matter (A). In the injured nongrafted animals, CGRP fibers were found in abundance in the dorsal aspect of the spinal cord near the lesion interface (B). Asterisk indicates lesion cavity. In β-gal transplants, CGRP fibers were limited within the graft, even in dorsal regions where CGRP fibers were sprouting in response to the injury. As shown in C (a BDNF graft,TP), growth factor-producing grafts contained large numbers of small-diameter, CGRP-ir fibers. These fibers appeared to make up the majority of small-diameter axons in the dorsal regions of the grafts.Arrows indicate host/graft interface in C.CHAT, Horizontal sections labeled for ChAT-ir from a control, uninjured spinal cord (D, 20×), an injured nongrafted spinal cord (E, 10×), a β-gal transplant (F, 20×), and an NT-3 graft (G, 20×). Note the lack of fiber staining in the injured, nongrafted spinal cord in and around the lesion (E; asterisk indicates lesion cavity). Some neurons appeared to have decreased ChAT levels caudal to the lesion. Grafts producing β-gal did not stimulate sprouting of ChAT-ir axons either adjacent to or within the grafts (F). Arrows indicate the interface between graft (TP) and host in F. Large-caliber ChAT-ir fibers extended from the host tissue into NT-3 (and BDNF) grafts (G). Serotonin (5-HT), Immunohistochemistry of a control uninjured spinal cord (H, 20×) and a β-gal-producing fibroblast graft (I, 10×, andJ, 40×; TP). 5-HT staining was restricted to small-diameter, varicose fibers within the spinal gray matter. Sparse numbers of 5-HT axons were noted in all transplants, including β-gal controls. Arrows indicate the interface between graft and host; arrowheads denote fibers crossing the graft/host border.

Fig. 5.

Neurofilament and MBP double-label immunohistochemistry. Coronal sections through epicenters from β-gal- (A), bFGF- (B), and NT-3-producing grafts (C, 5×) that are double-labeled for neurofilament (red) and MBP (green). White arrows delineate graft borders. Regions in white boxesare shown at high power (40×) below (D, E, F, respectively). Note that neurofilament-positive axons extended into β-gal and bFGF grafts (although to a lesser degree than in NT-3 or BDNF grafts; see Fig. 3D). These fibers, however, were only occasionally myelinated. In addition, the outer rim of host white matter appeared dysmyelinated in spinal cords that received bFGF grafts. Fibers entering NT-3 grafts were almost entirely myelinated as seen by the extensive numbers of axons (red) surrounded by myelin (green). As seen in G (enlargement of rectangle in F), neurofilament and MBP antibodies labeled separate entities (120×). Axons (white arrows, top panel) and lumens of myelin sheaths (white arrows, middle panel) are easily visible. When these images are merged, it is clear that the axons (red) were surrounded by the myelin sheaths (green).

Fig. 6.

Quantitation of the cross-sectional area occupied by MBP-positive profiles within the grafts and lesion epicenter. Several of the grafts, including β-gal, NT-3, BDNF, and CNTF, contained significantly more myelinated profiles than that observed in the lesion site of nontransplanted rats (Injury;^†p < 0.001). In addition, NT-3 and BDNF grafts contained significantly more MBP-positive fibers compared with all other grafts (*p < 0.001). MBP expression within CNTF, FGF, and NGF grafts was not different from β-gal grafts. (Bars represent mean ± SEM; n = 5 for injury control, NGF, CNTF, FGF; n = 6 for β-gal, NT-3;n = 11 for BDNF).

Fig. 7.

Comparison of Schwann cell and oligodendrocyte myelin within the grafts. Epicenter sections of an NGF-, bFGF-, or NT-3-producing graft at 70 d after injury (20×). Adjacent sections were stained for oligodendrocytes and their myelin sheaths (RIP, red; top panels) or Schwann cell myelin (P0,green; bottom panels). White lines delineate the graft (above) from host ventrolateral white matter (below). Note that NGF grafts (and CNTF and β-gal grafts; data not shown) appeared to have similar amounts of RIP- and P0-ir myelin, whereas bFGF grafts had minimal RIP-positive myelin but extensive Schwann cell-derived myelin. NT-3 grafts and (BDNF grafts; data not shown) had numerous RIP-ir cells and myelin with much less P0-ir myelin. In adjacent sections, P0 and RIP did not appear to overlap (arrowheads).

Fig. 8.

BrdU immunolabeling reveals increased numbers of mitotically active cells in general, and oligodendrocytes in particular, in NT-3 and BDNF grafts. A, Low-power photomicrographs (10×) of BrdU-labeled cells within the designated grafts demonstrate that the relative number of dividing cells was increased in BDNF and NT-3 grafts. B, High-power photomicrograph (320×) of an NT-3 graft demonstrates that many dividing cells (BrdU-positive) were double-labeled with RIP, revealing oligodendrocytes that were derived from cells that had undergone mitosis. When the RIP image is superimposed on the BrdU image, three BrdU-positive oligodendrocytes are visible (arrowheads). Note that these new oligodendrocytes were closely associated with myelin profiles.

Fig. 9.

Quantification of mitotically active cells within NT-3, BDNF, and β-gal grafts. A, Quantification of proliferating cells (Fig. 8A) revealed that the density of BrdU-positive cells was significantly greater in NT-3 and BDNF grafts compared with control grafts (***p < 0.001). In addition, NT-3 grafts contained significantly more proliferating cells than BDNF grafts (^†p< 0.001). B, Counts of RIP/BrdU double-labeled cells revealed that both NT-3 and BDNF grafts contained significantly more BrdU-positive oligodendrocytes than β-gal grafts (**p< 0.01, ***p < 0.001), whereas the number of BrdU-labeled Schwann cells and astrocytes (BrdU/S100β) was not different (C). D, The number of unidentified BrdU-positive cells (not colabeled with RIP or S100β) was significantly greater in NT-3 and BDNF grafts and may represent, at least in part, turnover of the fibroblast population. (Bars represent mean ± SEM; n = 5 for β-gal, NT-3;n = 4 for BDNF).