Astrocytes derived from glial-restricted precursors promote spinal cord repair

Abstract

Background: Transplantation of embryonic stem or neural progenitor cells is an attractive strategy for repair of the injured central nervous system. Transplantation of these cells alone to acute spinal cord injuries has not, however, resulted in robust axon regeneration beyond the sites of injury. This may be due to progenitors differentiating to cell types that support axon growth poorly and/or their inability to modify the inhibitory environment of adult central nervous system (CNS) injuries. We reasoned therefore that pre-differentiation of embryonic neural precursors to astrocytes, which are thought to support axon growth in the injured immature CNS, would be more beneficial for CNS repair.

Results: Transplantation of astrocytes derived from embryonic glial-restricted precursors (GRPs) promoted robust axon growth and restoration of locomotor function after acute transection injuries of the adult rat spinal cord. Transplantation of GRP-derived astrocytes (GDAs) into dorsal column injuries promoted growth of over 60% of ascending dorsal column axons into the centers of the lesions, with 66% of these axons extending beyond the injury sites. Grid-walk analysis of GDA-transplanted rats with rubrospinal tract injuries revealed significant improvements in locomotor function. GDA transplantation also induced a striking realignment of injured tissue, suppressed initial scarring and rescued axotomized CNS neurons with cut axons from atrophy. In sharp contrast, undifferentiated GRPs failed to suppress scar formation or support axon growth and locomotor recovery.

Conclusion: Pre-differentiation of glial precursors into GDAs before transplantation into spinal cord injuries leads to significantly improved outcomes over precursor cell transplantation, providing both a novel strategy and a highly effective new cell type for repairing CNS injuries.

Figure 1

The models of spinal cord injury in adult rats used in this study. Schematic illustrations of (a-c) white matter of the dorsal column and (d) the dorsolateral funiculus white matter pathways of the spinal cord. (a,d) Dorsal views of the rat brain and spinal cord. (b) horizontal and (c) sagittal views of the dorsal column white matter pathways at the C1/C2 cervical vertebrae of the spinal cord. (a) Dorsal column white matter on the right side was transected (shaded area) at the C1/C2 spinal level, and the ability of either BDA-labeled endogenous axons or axons from microtransplanted GFP-expressing adult sensory neurons (DRGs) to cross injuries bridged with GDAs or GRPs was assayed. (b) Injections of GDA or GRP cells (black diamonds) suspended in medium were made directly into the centers of the injury sites as well as their rostral and caudal margins in the cervical spinal cord. (c) A discreet population of endogenous ascending axons within the cuneate and gracile white matter pathways of dorsal columns was labeled by BDA injection at the C3/C4 spinal level (5 mm caudal to the lesion site, shaded). Alternatively, microtransplants of GFP⁺ DRGs were injected 500 μm caudal to the injury site. (d) The right-side dorsolateral funiculus white matter containing descending axons of the rubrospinal tract was transected at the C3/C4 spinal level and GDAs or GRPs were transplanted as described for dorsal column injuries. To trace axotomized rubrospinal tract axons, BDA was injected into the left-side red nucleus (RN) 8 days before the end of each experiment. CC, central canal; Cf, cuneate fasciculus; CST, corticospinal tract; DF, dorsolateral funiculus; Gf, gracile fasciculus; GM, gray matter; RST, rubrospinal tract; T1, level of the first thoracic vertebra.

Figure 2

Quantification of numbers of regenerating BDA⁺ axons in GDA-transplanted versus control dorsal column white matter at 8 days after injury and transplantation. BDA-labeled axons were counted in every third sagittally oriented section within the lesion center and at points 0.5 mm, 1.5 mm, and 5 mm rostral to the injury site, up to and including the dorsal column nuclei (DCN). Note that 61% of BDA⁺ axons had reached the centers of GDA-transplanted lesions and 39% to 0.5 mm beyond injury sites, compared with just 4% (lesion center) and 3.8% (0.5 mm rostral) present in controls. The steady decline in numbers of BDA⁺ axons within rostral white matter indicates a staggered front of maximum axon growth beyond sites of injury in GDA-transplanted groups at this time point. Note the total absence of axons at 5.0 mm rostral and in dorsal column nuclei in controls. Counts of BDA⁺ axons labeled in all adjacent sagittally oriented sections in representative GDA-treated and control lesioned cords revealed totals of 372 and 330 axons, respectively, at 0.5 mm caudal to the injury site. Increases in numbers of BDA⁺ axons in GDA-treated animals compared with controls were statistically significant (p < 0.01) in all rostral spinal cord regions. Error bars indicate ± 1 standard deviation.

Figure 3

Endogenous sensory axon regeneration across GDA-transplanted dorsal column injuries at 8 days after lesion and transplantation. (a) A montaged, low-magnification confocal image scanned from a single 25-μm thick sagittal section, showing BDA-labeled ascending dorsal column axons (green) that have entered, grown within and exited a hPAP⁺ (red) GDA-transplanted dorsal column lesion. LC, lesion center. (b) A high-magnification image of a rostral graft/host interface showing BDA⁺ axons exiting the GDA graft and entering host white matter. A few axons were observed to have turned away from the interface and grown back towards the lesion center (arrowhead). (c) In control lesions, the vast majority of BDA⁺ axons have formed dystrophic endings and failed to leave the caudal margins of the lesion, marked by hypertrophic GFAP⁺ astrocytes (red). (d) A high-magnification image showing numerous BDA⁺ axons that have successfully crossed the host/graft interface at the caudal lesion margin. A few cut axons (arrowheads) have, however, failed to leave the caudal lesion interface and can be seen to have turned and/or formed dystrophic endings, particularly in regions containing few hPAP⁺ GDAs (red). (e) BDA⁺ axons located near the pial surface and ventral regions of cuneate white matter at 1.5 mm rostral to a GDA-bridged lesion site. (f) BDA⁺ axon growth cones in white matter 1.5 mm rostral to the lesion site often display streamlined growth cones indicative of rapid growth. Scale bars: (a,c) 100 μm; (b-e) 50 μm; (f) 5 μm (top) and 10 μm (bottom).

Figure 4

A comparison of the ability of GDA versus GRP transplants to promote axon growth across dorsal column injuries from adjacent microtransplanted adult sensory neurons at 8 days after injury and transplantation. (a) A montaged, confocal image scanned from a single 75-μm thick sagittally oriented section showing GFP⁺ axons (green) entering and exiting a dorsal column lesion bridged with hPAP⁺(red) GDAs. (b) In two cases in which GDA transplants did not adequately fill the injury site or migrate into lesion margins, GFP⁺sensory axons failed to cross the caudal lesion margin and instead formed dystrophic endings identical to those in control untreated injuries. LC, lesion center. (c) Confocal montage showing the complete failure of transplanted GRPs to support the growth of GFP axons across a dorsal column injury. Note that, despite the ability of transplanted GRPs to span the injury site, the majority of GFP⁺ axons have formed dystrophic endings within the caudal lesion margin. Scale bars: (a) 300 μm; (b) 100 μm; (c) 200 μm.

Figure 5

A comparison of GFP⁺ axon and host astrocyte alignment in GDA- versus GRP- transplanted lesion margins at 8 days after injury. (a) A high-magnification image showing aligned axon growth (green) associated with aligned GFAP⁺ host astrocytic processes (red) in the caudal margin of a GDA-transplanted lesion. (b) In contrast, GFAP⁺ astrocytic processes (green) are misaligned in the caudal margin of a GRP-transplanted lesion (red). (c) A high-power confocal image showing GFP⁺ axons displaying tortuous, misaligned patterns of growth and dystrophic end bulbs (arrowhead) within the astrogliotic caudal margin of a GRP-transplanted lesion. Scale bars: (a) 25 μm; (b,c) 50 μm.

Figure 6

Reorganization of lesion margins by GDAs. (a,c) Control lesions; (b,d) transplanted lesions. Control lesions at (a) 4 days and particularly at (c) 8 days after injury have a dense meshwork of hypertrophic cell bodies and processes of endogenous astrocytes within lesion margins that is typical of forming glial scar tissue. (b) At 4 days after injury and transplantation, 'flares' of hPAP⁺ GDAs (green) are interwoven with realigned host GFAP⁺ astrocytes within lesion margins (the caudal margin is shown). Processes of both transplanted GDAs and host astrocytes are oriented towards the lesion center. Note that hPAP⁺ GDAs are not GFAP⁺. (d) At 8 days after injury and transplantation, GDAs have effected a reduction in host astrogliosis and a striking realignment of host GFAP⁺ astrocytes compared with the control (c). (e) Quantification of the alignment of host GFAP⁺ processes in lesion margins. The angles measured between each pair of GFAP⁺ processes in control (n = 100) and GDA-transplanted lesion margins (n = 100) are graphically displayed in a histogram. Each bin along the x-axis represents the angle between a pair of processes: 0° is parallel and 90° is perpendicular. The y-axis indicates the number of pairs of GFAP⁺ processes within each bin. Note the striking difference in alignment of GFAP⁺ host astrocytic processes in margins of GDA-transplanted lesions versus controls. GDA-transplanted lesions have an average angle of just 11.6° (median 7°) between paired processes, versus 59.4° (median 61°) for control lesion margins. Statistical analysis: p < 0.0001, t-test. Scale bars: (a,c,d) 100 μm; (b) 50 μm.

Figure 7

GDA transplantation suppresses neurocan and NG2 immunoreactivity. (a) At 4 days after injury, control lesion margins display dense neurocan immunoreactivity (green) mainly associated with fine, GFAP^- processes and to a lesser extent with GFAP⁺ astrocyte cell bodies (red). (b) Neurocan immunoreactivity at 4 days after injury and transplantation is greatly reduced in margins of hPAP⁺ GDA-transplanted lesions. (c) At 8 days after injury and GDA transplantation, neurocan immunoreactivity within lesion margins has increased compared with the 4-day time point. Note, however, that intra-lesion hPAP⁺ GDAs continue not to be immunoreactive to neurocan. (d-f) GDA-transplanted lesion centers (e,f) at 4 days after injury show a marked reduction in NG2 immunoreactivity (red) compared with (d) control lesions. hPAP⁺ cells are stained green. (g-i) Although overall NG2 immunoreactivity has increased within the center of GDA-transplanted lesions (h,i) at 8 days after injury compared with (e,f) the 4-day time point, it is reduced compared with the more uniformly distributed NG2 immunoreactivity within the center of control lesions at 8 days after injury. Scale bars: (a,b,g) 100 μm; (c,h,i) 50 μm; (d-f) 200 μm.

Figure 8

Transplanted GDAs promote regeneration of rubrospinal axons. (a) Confocal montage scanned through a depth of 60 μm, showing a small population of BDA⁺ rubrospinal tract (RST) axons (green) that have traversed a GDA-bridged (red) lesion of the dorsolateral funiculus and entered caudal white matter at 8 days after injury. The majority of RST axons, however, have sprouted to within 300 μm of the lesion center (LC) but failed to extend beyond the site of injury. Note the absence of BDA-labeled axons within the dorsal-most regions of the injury site. (b) Confocal montage showing the complete failure of axotomized BDA⁺ RST axons to cross control lesions at 8 days after injury and that the majority of axons have remained within rostral lesion margins at a distance of 500–800 μm from the lesion center (LC). (c) At 5 weeks after injury and transplantation, a small population of BDA⁺ RST axons have traversed GDA-bridged injury sites and extended within caudal white matter. Note that BDA⁺ axons have also sprouted into the dorsal regions of the lesion center and even extended beyond the pial surface (arrowhead; see also the high-power image in (d)). Note the lower levels of GFAP immunoreactivity (red) in more ventral regions of the injury margins and center, coincident with the presence of BDA⁺ axons. (e) Two examples of RST axons displaying growth cones within white matter 2 mm caudal to a GDA-treated lesion, at 5 weeks after transplantation. Note the collateral branch (asterisk). (f) Confocal image of a BDA⁺ terminal field-like axonal plexus within layer 5 spinal cord gray matter, immediately adjacent to the dorsolateral funiculus white matter at 5 weeks after injury and transplantation. In contrast, in all GDA-transplanted rats and controls injected with medium alone at 8 days after injury, no BDA labeling was observed within gray matter beyond the injury site. Scale bars: (a-c) 200 μm; (d) 100 μm; (e) 5 μm; (f) 10 μm.

Figure 9

GDA transplantation suppresses atrophy of red nucleus neurons and promotes robust behavioral recovery. (a) Injured left-side red nuclei in control rats contained an average of 52% of the neurons counted in uninjured right-side red nuclei at 5 weeks after injury. The numbers of neurons in the injured left-side red nuclei of GDA-transplanted animals, however, was 81% of total neuron numbers in uninjured right-side nuclei (*p < 0.01). (b) Grid-walk analysis of locomotor recovery. Graph showing the average number of mistakes per experimental group at different time points after injury for GDA-transplanted rats versus the control-lesion and sham-operated groups. GDA-transplanted animals (green) performed significantly better than lesioned controls at all post-injury time points (p < 0.05). (c) Transplanted GRPs do not promote locomotor recovery. Graph showing the average number of grid-walk mistakes per experimental group from 1 day before injury (baseline pre-lesion) to 2 weeks after injury for a separate series of matched RST-lesioned rats that received either GRP or GDA transplants versus lesion-only control rats. Note the complete failure of locomotor recovery in GRP-transplanted animals compared with lesion-only controls at all time points and confirmation of significant locomotor recovery in response to GDA transplantation (p < 0.05). cs, cyclosporine.

Glossary

Glossary