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. 2002 Mar 5;99(5):3246-51.
doi: 10.1073/pnas.052308899. Epub 2002 Feb 26.

Survival and regeneration of rubrospinal neurons 1 year after spinal cord injury

Brian K Kwon  1 , Jie Liu, Corrie Messerer, Nao R Kobayashi, John McGraw, Loren Oschipok, Wolfram Tetzlaff
Affiliations

Survival and regeneration of rubrospinal neurons 1 year after spinal cord injury

Brian K Kwon et al. Proc Natl Acad Sci U S A. .

Abstract

Scientific interest to find a treatment for spinal cord injuries has led to the development of numerous experimental strategies to promote axonal regeneration across the spinal cord injury site. Although these strategies have been developed in acute injury paradigms and hold promise for individuals with spinal cord injuries in the future, little is known about their applicability for the vast majority of paralyzed individuals whose injury occurred long ago and who are considered to have a chronic injury. Some studies have shown that the effectiveness of these approaches diminishes dramatically within weeks after injury. Here we investigated the regenerative capacity of rat rubrospinal neurons whose axons were cut in the cervical spinal cord 1 year before. Contrary to earlier reports, we found that rubrospinal neurons do not die after axotomy but, rather, they undergo massive atrophy that can be reversed by applying brain-derived neurotrophic factor to the cell bodies in the midbrain. This administration of neurotrophic factor to the cell body resulted in increased expression of growth-associated protein-43 and Talpha1 tubulin, genes thought to be related to axonal regeneration. This treatment promoted the regeneration of these chronically injured rubrospinal axons into peripheral nerve transplants engrafted at the spinal cord injury site. This outcome is a demonstration of the regenerative capacity of spinal cord projection neurons a full year after axotomy.

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Figures

Figure 1
Figure 1
Atrophy of rubrospinal neurons can be reversed by BDNF 6 and 12 months after injury. NeuN immunohistochemistry (a, b) (animals injured 12 months before) and cresol violet staining (c, d) (animals injured 6 months before) both demonstrate numerous atrophic neurons on the injured side treated with vehicle alone. Note the recovery in cell size of the BDNF-treated injured neurons to almost normal size as seen on the contralateral side in both NeuN (b) and cresyl violet staining (d). All sections are taken from comparable areas of the rubrospinal nucleus, approximately 240 μm from the caudal pole. (Bar = 50 μm.)
Figure 2
Figure 2
NeuN immunostaining specifically labels neurons and does not label astrocytes or microglia. (a) NeuN (green) and GFAP (red) immunohistochemistry for neurons and astrocytes, respectively, shows no overlap of labeling. (b) NeuN (green) and isolectin B4 (red) immunohistochemistry for neurons and microglia, respectively, shows no overlap of labeling. (Bar = 50 μm.)
Figure 3
Figure 3
Neuronal counts and cell cross-sectional area measurements. (a) The number of injured neurons is represented as a percentage of the number of contralateral uninjured neurons. Note that with BDNF treatment, the number of injured neurons counted is approximately 100% of the uninjured, in both NeuN and cresyl violet staining. With cresyl violet staining only, 89% of the number of uninjured neurons was detected in the vehicle treatment group. For the counts of uninjured neurons, 100% was 870 ± 68 neurons. (b) Histogram of cross-sectional area plotting neurons in 100-μm2 increments demonstrates a normalization of the distribution of cell sizes with the BDNF treatment. Note the predominance of small neurons in the vehicle-treated group. (Bar = 50 μm.)
Figure 4
Figure 4
In situ hybridization signals for GAP-43 and Tα1 tubulin mRNA. (a) At 12 months after injury, the GAP-43 signal in the injured vehicle-treated neurons is similar to background levels on the contralateral uninjured side. (b) The GAP-43 signal is greatly enhanced by BDNF treatment initiated 12 months after initial injury, an effect which is still achievable even 18 months after the injury (c). (d) Tα1 tubulin signal is low in injured neurons 12 months after axotomy, but is restored by BDNF treatment (e). (Bar = 50 μm.)
Figure 5
Figure 5
Full-length trkB receptor immunohistochemistry. (a) Axotomized rubrospinal neurons treated with vehicle solution only are very atrophic, 12 months after injury, yet maintain immunoreactivity for full-length trkB. (b) Axotomized rubrospinal neurons treated with BDNF 12 months after injury also maintain full-length trkB expression. Note the hypertrophy of some of the BDNF-treated rubrospinal neurons. (Bar = 50 μm.)
Figure 6
Figure 6
Regeneration of rubrospinal neurons 12 months after cervical spinal cord injury. (a) Schematic of transplantation procedures. (b) Labeling with DiI and fast blue shows single-labeled cells (arrow heads) and double-labeled cells (arrows). (c) Fast blue labeling of the same section as in b. (d) BDA labeling of regenerating rubrospinal neurons. (e) Same section as in d, demonstrating quenching/washout of the fast blue label caused by the BDA procedure. With BDA staining, double-labeled neurons (arrows) were still visible by an obvious halo of fast blue, with examples in f and g. Surrounding glial cells take up the washed-out fast blue (arrowhead). (h–j) Sagittal sections at the interface between host spinal cord and peripheral nerve graft demonstrate large BDA-labeled axons (arrows) in the position of the rubrospinal tract, which is consistent with the robust regenerative response in this BDNF-treated animal. (Bar = 50 μm.)
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