Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord

Abstract

We have recently reported that minimally disturbed adult CNS white matter can support regeneration of adult axons by using a novel microtransplantation technique to inject minute volumes of dissociated adult rat dorsal root ganglion neurons directly into adult rat CNS pathways (Davies et al., 1997). This atraumatic injection procedure minimized scarring and allowed considerable numbers of regenerating adult axons immediate access to the adult CNS glial terrain where they rapidly extended for long distances. A critical question remained as to whether degenerating white matter at acute and chronic stages (up to 3 months) after injury could still support regeneration. To investigate this, we have microtransplanted adult sensory neurons into degenerating white matter of the adult rat spinal cord several millimeters rostral to a severe lesion of the dorsal columns. Regeneration of donor sensory axons in both directions away from the site of transplantation was robust even within white matter undergoing fulminant Wallerian degeneration despite intimate contact with myelin. Along their route, the regrowing axons extended large numbers of collaterals into the adjacent dorsal horn. However, after entering the lesion, the rapidly extending growth cones stopped and became dystrophic within high concentrations of reactive glial matrix. Our results offer compelling evidence that the major environmental impediment to regeneration in the adult CNS is the molecular barrier that forms directly at the lesion site, and that degenerating white matter beyond the glial scar has a far greater intrinsic ability to support axon regeneration than previously thought possible.

Fig. 1.

Sites of transplantation and lesions. A photomicrograph of the hindbrain and cervical spinal cord of an adult rat that shows the relative positions of transplants and lesions in experimental sets 1–3 within the right side of the dorsal column sensory pathways. For experimental set 1, transplants were injected at the cervical level of C1/C2, and lesions were placed ∼14 mm caudally at the C4/C5 intervertebral junction. For experimental sets 2 and 3, transplants were injected into dorsal column white matter at the level of the medulla (asterisk), and lesions were placed 4–5 mm caudally at C1/C2 (visible in this particular animal). A portion of the cerebellum has been removed during dissection to expose the dorsal column nuclei (DCNs, arrows).

Fig. 2.

Long distance adult axon regeneration in degenerating spinal cord white matter. a, A confocal montage of scans from a single sagittally orientated 60 μm section showing the long distance growth (7.5 mm shown in this section) of numerous GFP-immunolabeled adult sensory axons that have extended caudally from a C1/C2 set 1 transplant (at 0 mm) within acutely lesioned dorsal column white matter at 8 d survival.b, A similarly constructed confocal montage showing the robust bidirectional regeneration of GFP+ donor axons from a set 1 transplant (Tp, arrowhead) injected into 2 week subchronically degenerated dorsal column white matter at 8 d survival after transplantation. Scale bar, 500 μm.c, A high-power confocal image of the transplant inb containing individual large and small neuronal cell bodies with their axons extending out into host white matter.Arrows indicate position of dorsal horn gray–cuneate white matter boundary. Scale bar, 200 μm. d, A double-channel confocal image of a coronally orientated 60 μm section immunostained for GFP (green channel) and GFAP (red channel) 2 mm rostral to an 8 d survival transplant at C1/C2. Numerous end on profiles of GFP+ regenerating axons can be seen throughout subchronically degenerating white matter of the cuneate (C,arrowhead) and gracile (G,arrowhead) fasciculi. M(arrowhead), Midline; Py, pyramidal tract; CC, central canal. Scale bar, 250 μm.e, A 1 μm semithin section cut in the coronal plane counterstained with toluidine blue of a representative C4/C5 lesion site. Note the complete transection of the cuneate fasciculus (C, arrowhead) and sparing of only a small portion of the most medial axons of the gracile tract (G, arrowhead). Scale bar, 200 μm.

Fig. 3.

Terminal field invasion.a, A confocal montage of scans from a single sagittally orientated 60 μm section showing substantial numbers of primary GFP+ axons (green channel) growing at all points dorsoventrally within 2 week subchronically degenerating dorsal column (DC) white matter that are either sending rectilinear collateral branches or turning at right angles themselves to invade and ramify within the host dorsal horn (DH) gray matter (GFAP red channel). b, A section ofa at higher power in which the GFAP staining within gray matter (within the asterisk-demarcated box) has been digitally intensified to show the relationship of the rectilinear fascicles formed by some invading GFP+ axons with the gray matter astrocytes of the dorsal horn. Graft survival, 8 d. Scale bars: a, 250; b, 100 μm.

Fig. 4.

Morphology of growth cones and relationship to host astrocytes. a, A confocal scan at a distance of 4 mm caudal to an 8 d survival set 1 transplant showing GFP+ (green channel) axons growing in parallel to the longitudinal processes of host astrocytes (GFAP, red channel) within acutely degenerating white matter at its interface with host dorsal horn gray matter that contains typically stellate astrocytes. Arrow 1 indicates an example of a growth cone at the gray–white interface (higher power panel b) displaying several filopodia on a more expanded tip, possibly indicating that it is in the process of making a decision to invade gray matter. Arrow 2 points to an example of a “streamlined” growth cone with a single filopodium, a morphology typically displayed by regenerating axons within white matter. c shows this growth cone at higher power as it skirts a blood vessel. Scale bars: a, 50 μm; b, c, 10 μm.

Fig. 5.

Relationship of growing axons to white matter myelin and astrocytes. a, b, Electron microscope images of GFP-immunolabeled regenerating adult DRG axons in intimate contact (arrowheads) with degenerating myelin membranes within acutely lesioned dorsal column white matter. Three day survival after transplantation and 1.5 mm distance from the graft ensures that these profiles are near the growing tips of the axons. However, the dense HRP labeling prevented the ability to distinguish bona fide growth cones from axonal varicosities. Note that the GFP-immunolabeled axon profile in a is also tightly associated with an adjacent host astrocyte (As). Magnification 5000×.

Fig. 6.

Failure of regeneration on reaching the glial scar. a, b, Double channel confocal images (60 μm) from two 8 d survival set 2 cases showing numerous caudally regenerating GFP+ (green channel) adult sensory axons that have grown 4–5 mm through acutely degenerating and gliotic (GFAP, red channel) dorsal column white matter to eventually invade and stop within the lesion sites (L,arrowhead). c, d, Triple channel confocal images of the same lesion sites in aand b (a, d;b, c) that are additionally stained for CSPGs (blue channel) and show that the failure of regeneration correlates with axons entering high levels of inhibitory proteoglycans found only within the lesion site. e, A high-power triple channel confocal scan (GFP, green; GFAP, red; CSPG, blue) showing that the growth cones of regenerating GFP+ axons have been transformed into dystrophic endings within the CSPG-rich, reactive astrocytic terrain of the lesion. Scale bars: a, b, 200 μm;c, d, 250 μm; e, 25 μm.

Fig. 7.

Axon regeneration in chronic degenerating white matter and its failure after reaching the glial scar. a,b, Double and triple channel confocal images, respectively (GFP, green channel; GFAP, red channel; CSPG, blue channel) through 60 μm of a lesion site from 3 month chronic set 3 animal that has received an adult DRG neuron microtransplant rostral to the injury site. Numerous GFP+ axons have regenerated 4–5 mm caudally from the site of transplantation through chronically degenerating dorsal column white matter. After reaching mature scar, the regenerating axon tips have stopped and taken on a dystrophic appearance at varying distances from the center of the lesion (b,asterisks) within the CSPG (b,blue channel)-immunoreactive and intensely GFAP+-reactive gliotic scar tissue bordering the site of injury. Note the abrupt termination of regeneration at the center of the lesion site for the few more ventrally placed axons that were capable of traversing the correspondingly shorter rostral spread of reactive astroglia compared with that found nearer the pial surface. Survival 8 d after transplantation. Scale bars, 250 μm.