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, 33 (13), 5655-67

A Novel Growth-Promoting Pathway Formed by GDNF-overexpressing Schwann Cells Promotes Propriospinal Axonal Regeneration, Synapse Formation, and Partial Recovery of Function After Spinal Cord Injury

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A Novel Growth-Promoting Pathway Formed by GDNF-overexpressing Schwann Cells Promotes Propriospinal Axonal Regeneration, Synapse Formation, and Partial Recovery of Function After Spinal Cord Injury

Ling-Xiao Deng et al. J Neurosci.

Abstract

Descending propriospinal neurons (DPSN) are known to establish functional relays for supraspinal signals, and they display a greater growth response after injury than do the long projecting axons. However, their regenerative response is still deficient due to their failure to depart from growth supportive cellular transplants back into the host spinal cord, which contains numerous impediments to axon growth. Here we report the construction of a continuous growth-promoting pathway in adult rats, formed by grafted Schwann cells overexpressing glial cell line-derived neurotrophic factor (GDNF). We demonstrate that such a growth-promoting pathway, extending from the axonal cut ends to the site of innervation in the distal spinal cord, promoted regeneration of DPSN axons through and beyond the lesion gap of a spinal cord hemisection. Within the distal host spinal cord, regenerated DPSN axons formed synapses with host neurons leading to the restoration of action potentials and partial recovery of function.

Figures

Figure 1.
Figure 1.
The majority of neurons whose axons extended into the distal host spinal cord originated from descending propriospinal neurons. A, Experimental time line. B, Schematic drawing displayed the distribution of FG retrogradely labeled propriospinal neurons. Most labeled neurons were located in the intermediate gray matter and medial portion of the ventral horn (laminae V–VIII) between the T7–T10 cord segments. C, Bar graph shows that significantly more FG-labeled neurons were found in the group when SCs-GDNF was injected into the caudal spinal cord to form a continuous growth-promoting pathway compared with groups with injections of either SCs-GFP or DMEM at these spinal levels (***p < 0.001).
Figure 2.
Figure 2.
Descending propriospinal axons regenerated across the caudal graft–host interface and grew back into the distal host spinal cord. A, Schematic drawing shows the experimental strategy and how tissue was sampled. Note that labeled axons descending down to the contralateral hemicord were stopped at the rostral edge of a complete transection of the hemicord made right before the tracer injection. B, On the ipsilateral side of the bridge transplantation, BDA (green) anterogradely labeled propriospinal axons (white arrows) were found to penetrate through the distal graft–host interface (white dashed line) and to elongate within the distal host spinal cord only in the group injected with SCs-GDNF into the caudal host tissue. C, The distal graft–host interface was demarcated by GFAP-labeled astrocytes (red). D, Quantification of regenerated axonal numbers at different distances from the graft–host interface among three treatment groups. (***p < 0.001, **p < 0.01, *p < 0.05, compared with the distal SC-DMEM group; ▴▴▴p < 0.001, ▴▴p < 0.01, compared with the distal SC-GFP group). Scale bars: (in C) B, C, 100 μm.
Figure 3.
Figure 3.
Evidence for complete transection of the hemicord contralateral to the bridge transplantation. Schematic drawing illustrates the contralateral hemisection and how samples were taken. Three horizontal sections represent the dorsal (A), middle (B), and ventral (C) portions of the spinal cord. GFAP staining (green) outlines rostral and caudal boundaries of the transection (red dashed lines). Between the two boundaries, a clear complete transection of the contralateral hemicord (*) was seen. Dashed white lines outline the medial wall of the guidance channel.
Figure 4.
Figure 4.
Only BDA-labeled axons that regenerated through the bridge transplant re-enter the distal host spinal cord. A, Schematic drawing illustrates that BDA-labeled axons regenerate through and beyond a bridge transplant. In the contralateral hemicord, however, BDA-labeled axons are completely stopped after the transection of spared hemicord. B, A representative horizontal section shows that BDA-labeled axons grew through a regenerative tissue cable, penetrated the distal graft–host interface (yellow curved line), and re-entered the host spinal cord (yellow arrows). On the contralateral side, however, the labeled axons (red arrows) completely stopped at the site of hemicord transection (white curved line). C–E, representative cross sections taken from corresponding levels of the graft and adjacent cord tissue shown in B (white straight lines). C, Within the graft rostral to the contralateral transection, BDA-labeled axons (white arrows) were seen both within the graft and the contralateral hemicord. D, Within the graft caudal to the contralateral transection, BDA-labeled axons were found only within the graft (white arrow). E, In the host spinal cord caudal to the graft–host interface, BDA-labeled regenerated axons appeared only in the spinal cord ipsilateral to the graft. High magnification of the boxed area in E shows details of BDA-labeled regenerated axons in the distal host spinal cord. Arrows in B indicate the guidance channel wall. Yellow circles in C and D outline the guidance channel wall in cross sections. White dashed lines in C–E outline the gray matter areas. Scale bars: B, 400 μm; C–E, 500 μm.
Figure 5.
Figure 5.
Regenerated descending propriospinal axons formed new synapses on neurons/dendrites within the distal host spinal cord. A, BDA-anterogradely labeled descending propriospinal axons (red, arrows) grew through the distal graft–host interface (dashed line) back into the host spinal cord. The grafted guidance channel is outlined by yellow lines. B, C, High magnification of demarcated areas in A shows sites of new synapse formation (synaptophysin-IR, green) between regenerated axons (BDA-labeled, red) and host dendrites (MAP2-IR; blue). Synapse formation can be further appreciated in the upper left insert in C, which shows a high magnification of the boxed area in the same image in single section depicting the relationship between a regenerated axon and a few synaptic terminals on dendrites in XY, XZ, and YZ planes. The bottom right insert in C is an EM image showing a BDA-labeled presynaptic terminal (*) containing numerous synaptic vesicles. D, Schematic drawing plotted the distribution of regenerated axons (dots) in a cross section of the spinal cord at L1. Note that axons appeared in the gray matter of the intermediate and ventral horn areas as well as in the lateral white matter, although the majority was located in the gray matter. E, A low magnification of a representative transverse section of L1 spinal cord on the ipsilateral side of the injury showed that regenerated axons, anterogradely labeled with BDA (red), formed synaptic contacts on the dendritic profiles labeled by anti-synaptophysin (green). White dashed line outlines the gray matter. F, High magnification of boxed area in E shows colocalization of regenerated axons (BDA, red) with dendritic profiles (synaptophysin-IR, green), which can be further appreciated in a confocal image in an adjacent section (insert of F). Scale bars: A, E, F, 100 μm; B, C, 50 μm. EM, 500 nm.
Figure 6.
Figure 6.
Grafted SCs survived and facilitated axonal regeneration. A, Schematic drawing shows how tissue was sampled. Three cross sections caudal to the graft were cut sequentially in rostrocaudal orientation. B–J, Representative sections, correspondence to the sections illustrated in A, at distances proximal (B–D), further (E–G), and furthest (H–J) to the grafted channel. In the proximal and further sections, BDA-labeled regenerated axons (red) were present not only within the territory of GFP-SCs (green arrows in D and G) but also in the territory without GFP-SCs (red arrows in D and G). In the furthest section, BDA-labeled regenerated axons (red) only appeared in the territory of grafted GFP-SCs (green arrow in J). The close association between GFP-SCs and BDA-labeled regenerated axons could be further appreciated in the high-magnification inserts of box areas shown in D, G, and J.
Figure 7.
Figure 7.
Grafted SCs remyelinated regenerated axons in the distal host spinal cord. A–C, At 8 weeks after transplantation, regenerated axons (BDA labeled, red) were myelinated by SCs (P0; green, yellow arrows). D–G, Triple labeling of a cross section showed that BDA-labeled regenerated axons (red, F) were myelinated by grafted SCs (GFP, D) that expressed a peripheral myelin marker P0 (purple, E), which can be further appreciated in the merged image (G). H–K, Many grafted SCs (GFP; H) formed myelin (P0; purple) on host axons (NF, blue). Scale bar, 10 μm.
Figure 8.
Figure 8.
Extracellular recordings of field potentials in the spinal cord. Electrical stimuli were delivered with a bipolar stimulating electrode located within the spinal intermediate gray region of the rostral spinal cord, and field potentials were recorded in the caudal spinal cord (across the bridge transplant) on the ipsilateral side. Representative traces (A, C, E) and related schematic drawings (B, D, F) are demonstrated from the same rat that received SCs-GDNF bridge transplant followed by injections of SCs-GDNF gradient into the ipsilateral distal host spinal cord. A, B, Field potential was recorded with the contralateral hemicord remained intact. C, D, Field potential was recorded after the transection of the contralateral hemicord. E, F, Field potential was recorded after the transection of the bridge transplant at the rostral graft–host interface. Note that the signal was abolished after total transection. G, Quantitative data show the amplitude of maximal voltage response in four groups. SC-GDNF treatment group, at both before or after contralateral hemisection, produced a significant increase in amplitude (*p < 0.05) when compared with that of either vehicle or SC-GFP treatment group. Importantly, the SC-GDNF treatment-induced increase in amplitude could be almost completely abolished by the total transection (#p < 0.05) compared with the amplitude recorded before and after contralateral hemisection.
Figure 9.
Figure 9.
Partial recovery of function after axonal regeneration through a continuous SCs-GDNF growth-promoting pathway. A, Improved BBB locomotor recovery was found in the group that received caudal injections of SCs-GDNF (red), compared with caudal injections of SCs-GFP (blue) or DMEM (green) groups (***p < 0.001, SCs-GDNF vs SCs-GFP; ▴▴▴p < 0.001, SCs-GDNF vs DMEM group). The effect of recovery was diminished after contralateral hemisection at week 7 and was partially recovered only in the SCs-GDNF-injected group at week 8. B, Increased stride length on the graft side was found in the group that received caudal injections of SCs-GDNF (red bar) when compared with caudal injections of SCs-GFP (blue bar, ***p < 0.001) or DMEM (green bar, ▴▴p < 0.01). RSL, right stride length.

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