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Pharmacological Suppression of CNS Scarring by Deferoxamine Reduces Lesion Volume and Increases Regeneration in an In Vitro Model for Astroglial-Fibrotic Scarring and in Rat Spinal Cord Injury In Vivo.

Vogelaar CF, König B, Krafft S, Estrada V, Brazda N, Ziegler B, Faissner A, Müller HW - PLoS ONE (2015)

Bottom Line: DFO could be identified as a putative anti-scarring treatment for CNS trauma.We subsequently validated this by local application of DFO to a dorsal hemisection in the rat thoracic spinal cord.DFO treatment led to significant reduction of scarring, slightly increased regeneration of corticospinal tract as well as ascending CGRP-positive axons and moderately improved locomotion.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurobiology Laboratory, Department of Neurology, Heinrich-Heine-University of Duesseldorf, Duesseldorf, Germany; Institute of Microanatomy and Neurobiology, Johannes Gutenberg-University Mainz, Mainz, Germany.

ABSTRACT
Lesion-induced scarring is a major impediment for regeneration of injured axons in the central nervous system (CNS). The collagen-rich glial-fibrous scar contains numerous axon growth inhibitory factors forming a regeneration-barrier for axons. We demonstrated previously that the combination of the iron chelator 2,2'-bipyridine-5,5'-decarboxylic acid (BPY-DCA) and 8-Br-cyclic AMP (cAMP) inhibits scar formation and collagen deposition, leading to enhanced axon regeneration and partial functional recovery after spinal cord injury. While BPY-DCA is not a clinical drug, the clinically approved iron chelator deferoxamine mesylate (DFO) may be a suitable alternative for anti-scarring treatment (AST). In order to prove the scar-suppressing efficacy of DFO we modified a recently published in vitro model for CNS scarring. The model comprises a co-culture system of cerebral astrocytes and meningeal fibroblasts, which form scar-like clusters when stimulated with transforming growth factor-β (TGF-β). We studied the mechanisms of TGF-β-induced CNS scarring and compared the efficiency of different putative pharmacological scar-reducing treatments, including BPY-DCA, DFO and cAMP as well as combinations thereof. We observed modulation of TGF-β-induced scarring at the level of fibroblast proliferation and contraction as well as specific changes in the expression of extracellular matrix molecules and axon growth inhibitory proteins. The individual and combinatorial pharmacological treatments had distinct effects on the cellular and molecular aspects of in vitro scarring. DFO could be identified as a putative anti-scarring treatment for CNS trauma. We subsequently validated this by local application of DFO to a dorsal hemisection in the rat thoracic spinal cord. DFO treatment led to significant reduction of scarring, slightly increased regeneration of corticospinal tract as well as ascending CGRP-positive axons and moderately improved locomotion. We conclude that the in vitro model for CNS scarring is suitable for efficient pre-screening and identification of putative scar-suppressing agents prior to in vivo application and validation, thus saving costs, time and laboratory animals.

No MeSH data available.


Related in: MedlinePlus

Effects of scar-reducing treatments on protein expression. Effects of treatments on protein levels of ECM molecules.(A) Collagen I/III/V was detected by F1C3 antibody, which stained 5 bands corresponding to collagen polypeptide chains that contribute to the three collagen subtypes. (B) Tnc was detected with KAF14 antibody which stained two bands. (C) α-Tubulin served as a loading control. (D) The upper two collagen bands were reduced by DFO. The reduction in DFO + cAMP was exclusively due to DFO, since cAMP alone did not change collagen levels. (E) The upper band of Tnc was reduced by DFO, cAMP and combinations thereof. Statistics: one way Anova with Bonferroni post-hoc test * p < 0.05, ** p < 0.01, *** p< 0.001.
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pone.0134371.g006: Effects of scar-reducing treatments on protein expression. Effects of treatments on protein levels of ECM molecules.(A) Collagen I/III/V was detected by F1C3 antibody, which stained 5 bands corresponding to collagen polypeptide chains that contribute to the three collagen subtypes. (B) Tnc was detected with KAF14 antibody which stained two bands. (C) α-Tubulin served as a loading control. (D) The upper two collagen bands were reduced by DFO. The reduction in DFO + cAMP was exclusively due to DFO, since cAMP alone did not change collagen levels. (E) The upper band of Tnc was reduced by DFO, cAMP and combinations thereof. Statistics: one way Anova with Bonferroni post-hoc test * p < 0.05, ** p < 0.01, *** p< 0.001.

Mentions: The influence of the scar-suppressing treatments on the protein expression of the ECM molecules collagen and Tnc was studied in more detail using the F1C3 and KAF14 antibodies, respectively. The F1C3 antibody detected five bands in total of estimated 180, 210, 230 and >250 kDa in size, representing the various collagen polypeptide chains that are present in collagen I, III, and V [42]. DFO, alone or combined with cAMP, significantly reduced the levels of the upper two collagen bands (Fig 6A and 6D). Two Tnc protein bands were detected by KAF14, of estimated 230 and >250 kDa in size. The upper band was significantly reduced by DFO, cAMP and combinations thereof. (Fig 6B and 6E).


Pharmacological Suppression of CNS Scarring by Deferoxamine Reduces Lesion Volume and Increases Regeneration in an In Vitro Model for Astroglial-Fibrotic Scarring and in Rat Spinal Cord Injury In Vivo.

Vogelaar CF, König B, Krafft S, Estrada V, Brazda N, Ziegler B, Faissner A, Müller HW - PLoS ONE (2015)

Effects of scar-reducing treatments on protein expression. Effects of treatments on protein levels of ECM molecules.(A) Collagen I/III/V was detected by F1C3 antibody, which stained 5 bands corresponding to collagen polypeptide chains that contribute to the three collagen subtypes. (B) Tnc was detected with KAF14 antibody which stained two bands. (C) α-Tubulin served as a loading control. (D) The upper two collagen bands were reduced by DFO. The reduction in DFO + cAMP was exclusively due to DFO, since cAMP alone did not change collagen levels. (E) The upper band of Tnc was reduced by DFO, cAMP and combinations thereof. Statistics: one way Anova with Bonferroni post-hoc test * p < 0.05, ** p < 0.01, *** p< 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4519270&req=5

pone.0134371.g006: Effects of scar-reducing treatments on protein expression. Effects of treatments on protein levels of ECM molecules.(A) Collagen I/III/V was detected by F1C3 antibody, which stained 5 bands corresponding to collagen polypeptide chains that contribute to the three collagen subtypes. (B) Tnc was detected with KAF14 antibody which stained two bands. (C) α-Tubulin served as a loading control. (D) The upper two collagen bands were reduced by DFO. The reduction in DFO + cAMP was exclusively due to DFO, since cAMP alone did not change collagen levels. (E) The upper band of Tnc was reduced by DFO, cAMP and combinations thereof. Statistics: one way Anova with Bonferroni post-hoc test * p < 0.05, ** p < 0.01, *** p< 0.001.
Mentions: The influence of the scar-suppressing treatments on the protein expression of the ECM molecules collagen and Tnc was studied in more detail using the F1C3 and KAF14 antibodies, respectively. The F1C3 antibody detected five bands in total of estimated 180, 210, 230 and >250 kDa in size, representing the various collagen polypeptide chains that are present in collagen I, III, and V [42]. DFO, alone or combined with cAMP, significantly reduced the levels of the upper two collagen bands (Fig 6A and 6D). Two Tnc protein bands were detected by KAF14, of estimated 230 and >250 kDa in size. The upper band was significantly reduced by DFO, cAMP and combinations thereof. (Fig 6B and 6E).

Bottom Line: DFO could be identified as a putative anti-scarring treatment for CNS trauma.We subsequently validated this by local application of DFO to a dorsal hemisection in the rat thoracic spinal cord.DFO treatment led to significant reduction of scarring, slightly increased regeneration of corticospinal tract as well as ascending CGRP-positive axons and moderately improved locomotion.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurobiology Laboratory, Department of Neurology, Heinrich-Heine-University of Duesseldorf, Duesseldorf, Germany; Institute of Microanatomy and Neurobiology, Johannes Gutenberg-University Mainz, Mainz, Germany.

ABSTRACT
Lesion-induced scarring is a major impediment for regeneration of injured axons in the central nervous system (CNS). The collagen-rich glial-fibrous scar contains numerous axon growth inhibitory factors forming a regeneration-barrier for axons. We demonstrated previously that the combination of the iron chelator 2,2'-bipyridine-5,5'-decarboxylic acid (BPY-DCA) and 8-Br-cyclic AMP (cAMP) inhibits scar formation and collagen deposition, leading to enhanced axon regeneration and partial functional recovery after spinal cord injury. While BPY-DCA is not a clinical drug, the clinically approved iron chelator deferoxamine mesylate (DFO) may be a suitable alternative for anti-scarring treatment (AST). In order to prove the scar-suppressing efficacy of DFO we modified a recently published in vitro model for CNS scarring. The model comprises a co-culture system of cerebral astrocytes and meningeal fibroblasts, which form scar-like clusters when stimulated with transforming growth factor-β (TGF-β). We studied the mechanisms of TGF-β-induced CNS scarring and compared the efficiency of different putative pharmacological scar-reducing treatments, including BPY-DCA, DFO and cAMP as well as combinations thereof. We observed modulation of TGF-β-induced scarring at the level of fibroblast proliferation and contraction as well as specific changes in the expression of extracellular matrix molecules and axon growth inhibitory proteins. The individual and combinatorial pharmacological treatments had distinct effects on the cellular and molecular aspects of in vitro scarring. DFO could be identified as a putative anti-scarring treatment for CNS trauma. We subsequently validated this by local application of DFO to a dorsal hemisection in the rat thoracic spinal cord. DFO treatment led to significant reduction of scarring, slightly increased regeneration of corticospinal tract as well as ascending CGRP-positive axons and moderately improved locomotion. We conclude that the in vitro model for CNS scarring is suitable for efficient pre-screening and identification of putative scar-suppressing agents prior to in vivo application and validation, thus saving costs, time and laboratory animals.

No MeSH data available.


Related in: MedlinePlus