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Research Center for Cellular and Molecular Neurobiology, University of Liège, Liège, Belgium
1Correspondence: Research Center for Cellular and Molecular Neurobiology, University of Liège, Tour de Pathologie B36, 1erétage, local 1/4A, CHU Sart-Tilman 4000 Liège, Belgium. E-mail address: rfranzen{at}ulg.ac.be
SPECIFIC AIMS
The original aim of this study was to assess the effect of the stimulation of endogenous macrophages by the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) on axonal regrowth and locomotor recovery using a spinal cord injury model in adult rats. It was based on a previous study from our laboratory showing a phenotypic change of macrophages 3 wk postinjury, concomitant to an involution of spontaneous axonal regrowth.
PRINCIPAL FINDINGS
Boosting or modulating the immune response after spinal cord injury could be a promising strategy for a successful repair. Macrophages (monocytes/microglia) are thus considered to be key actors for a successful axonal regeneration. We therefore decided to treat paraplegic rats by one single delayed intraperitoneal injection of 2 µg of the cytokine GM-CSF, which is well known to be involved in macrophage activation.
1. GM-CSF improves locomotor performance
With the use of compression-lesion model, adult rat spinal cords were severely injured, resulting in an irreversible paraplegia. The locomotor performances of the rats were evaluated weekly by the use of the Basso, Beattie, Bresnahan open field locomotor test (BBB) during 24 wk. At the time of the treatment, i.e., 4 wk after the injury, rats were unable to move any of their hind-limb joints (mean BBB scores=0.86±0.2), demonstrating the extremely high severity of the lesion. As soon as 1 wk after the intraperitoneal injection, GM-CSF treated rats had a significantly higher mean BBB score (1.8±1.4), whereas the control group did not improve at all (0.69±1; P<0.05). The difference between the two groups remained obvious over the 10 subsequent wk, reaching the level of statistical significance at post-treatment weeks 4 and 7 (Fig. 1
). BBB scores between 4 and 6 were only reached by animals belonging to the GM-CSF group and not by those treated with saline. Scores of 4 to 6 indicate that the rats are able to move all three hind-limb joints slightly or extensively, whereas lower scores in the control group reflect at best slight motility at one or two joints.
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2. GM-CSF enhances fiber regeneration and brain-derived neurotrophic factor expression by endogenous macrophages
The spinal cord lesion model used in this study results in a subtotal destruction of both gray and white matter at the lesion epicenter. Twenty-four weeks postinjury, numerous longitudinally orientated growth-associated protein-43 immunoreactive fibers were detected within the lesion site of GM-CSF treated rats (Fig. 2
). This significant axonal regeneration was confirmed by the 200 kDa neurofilaments immunostaining. Interestingly, numerous serotonergic fibers were detected in the lesion site of GM-CSF treated spinal cords, whereas very few were seen in the control group. Image analysis of all three immunostainings revealed a significant difference between GM-CSF and saline-treated groups.
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To investigate the effect of GM-CSF injection on endogenous cell populations, the expression of the neurotrophin brain-derived neurotrophic factor (BDNF), well known for its favorable action on axonal regeneration, was assessed 1 wk after the treatment. We show that in GM-CSF treated group a large majority of endogenous macrophages express BDNF compared with the control group.
3. GM-CSF enhances neurite growth via an increased BDNF expression by microglial cells
We next examined BDNF protein expression in lysates of microglial cells cultured for 24 h in media containing or not recombinant GM-CSF (25 ng/ml). The expression of BDNF by microglia was higher when cells were activated with GM-CSF.
Adult dorsal root ganglion (DRG) neurons cocultured for 16 h with GM-CSF preactivated microglial cells were significantly more likely to bear TUJ-1 immunostained neurites than those cocultured with nonactivated cells (P<0.01). This increase in neuritogenesis of DRG neurons was abolished when an anti-BDNF antibody (1 µg/ml) was added to the cocultures.
CONCLUSION AND SIGNIFICANCE
This study demonstrates for the first time that a single intraperitoneal injection of 2 µg GM-CSF, a macrophage-activating cytokine, 4 wk after a spinal cord injury producing irreversible paraplegia in rats is able to promote locomotor recovery and axonal regrowth. It also shows that GM-CSF activated macrophages express more BDNF in vivo and in vitro and that they increase the neuritic index of cocultured neurons. Taken together, these results suggest that the favorable action of GM-CSF observed in experimental paraplegia could at least partially be mediated by the neurotrophic factor BDNF.
The findings of this study are important for the research field of paraplegia. They show that endogenous cells, i.e., macrophages, can be recruited to favor axonal regeneration and locomotor recovery. They are in complete accordance with other studies showing the beneficial role of macrophages in spinal cord injury. Already known to be favorable through their phagocytic activity, we show here that in addition macrophages can be recruited for their capacity to secrete neurotrophic factors known to support axonal regeneration. Moreover, our results open new perspectives and hopes for clinical application, as the strategy used to recruit and activate macrophages consists in a single delayed intraperitoneal injection of a cytokine already widely used in oncohematological disorder, without the need of any invasive intervention. Another advantage is that the treatment can be delayed in time and thus applied in larger patient groups, possibly also in chronic paraplegics.
Our results are also in accordance with a recent published work describing a favorable effect of GM-CSF on locomotor recovery in another less severe model of spinal cord injury in rats.
Finally, our study strongly suggests that all those beneficial effects could be at least partially mediated by the neurotrophin BDNF by increasing its secretion by GM-CSF stimulated macrophages. BDNF is known to stimulate locomotor activity after both moderate and severe spinal cord lesion in rats by activating the local central pattern generator and axonal growth notably via intraneuronal increase of cAMP. It also promotes the regenerative sprouting of injured serotonergic axons in the adult rat brain (Fig. 3
).
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Now that the beneficial effect of the cytokine GM-CSF on axonal regrowth and locomotor improvement is demonstrated, further studies are necessary to investigate the molecular mechanisms involved and, in the perspective of a clinical application, the effect of repeated injections and/or of the combination of this treatment with other regenerative strategies.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4382fje
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  S. H. Yoon, Y. S. Shim, Y. H. Park, J. K. Chung, J. H. Nam, M. O. Kim, H. C. Park, S. R. Park, B.-H. Min, E. Y. Kim, et al. Complete Spinal Cord Injury Treatment Using Autologous Bone Marrow Cell Transplantation and Bone Marrow Stimulation with Granulocyte Macrophage-Colony Stimulating Factor: Phase I/II Clinical Trial Stem Cells, August 1, 2007; 25(8): 2066 - 2073. [Abstract] [Full Text] [PDF]
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