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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 TGF-β and scar-reducing treatments on gene expression.(A) Effects of TGF-β on the mRNA expression of ECM molecules and axon growth inhibitors in cortical astrocytes and meningeal fibroblasts cultured separately and in the co-cultures. Levels of each target mRNA were normalized to the housekeeping gene cyclophilin. Statistics: unpaired T-test * p < 0.05, ** p < 0.01. (B) Effects of scar-reducing treatments. Plotted are the levels of target mRNAs normalized to cyclophilin relative to the H2O control treatment. DFO reduced the levels of collagen IV and NG-2. Treatment with cAMP led to a reduction in Tnc. Neurocan and phosphacan were upregulated in DFO + cAMP, whereas NG-2 was significantly reduced after DFO treatment and in DFO + cAMP co-treatments compared to cAMP alone. Statistics: one way Anova with Bonferroni * p < 0.05, ** p < 0.01, *** p< 0.001.
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pone.0134371.g004: Effects of TGF-β and scar-reducing treatments on gene expression.(A) Effects of TGF-β on the mRNA expression of ECM molecules and axon growth inhibitors in cortical astrocytes and meningeal fibroblasts cultured separately and in the co-cultures. Levels of each target mRNA were normalized to the housekeeping gene cyclophilin. Statistics: unpaired T-test * p < 0.05, ** p < 0.01. (B) Effects of scar-reducing treatments. Plotted are the levels of target mRNAs normalized to cyclophilin relative to the H2O control treatment. DFO reduced the levels of collagen IV and NG-2. Treatment with cAMP led to a reduction in Tnc. Neurocan and phosphacan were upregulated in DFO + cAMP, whereas NG-2 was significantly reduced after DFO treatment and in DFO + cAMP co-treatments compared to cAMP alone. Statistics: one way Anova with Bonferroni * p < 0.05, ** p < 0.01, *** p< 0.001.

Mentions: We studied the mRNA expression of various ECM molecules and axon growth inhibitors by real time quantitative PCR (Fig 4, Panel A). We extracted RNA from the astrocyte-fibroblast co-cultures as well as from the individual cell mono-cultures, treated with and without TGF-β, in order to investigate the relative contribution of the two cell types. We normalized to cyclophilin, a housekeeping gene that stayed constant after TGF treatment as opposed to GAPDH and ODC which were both regulated by TGF (data not shown). The relative expression levels of the molecules of interest allowed us to estimate their relative contribution to the scar matrix.


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 TGF-β and scar-reducing treatments on gene expression.(A) Effects of TGF-β on the mRNA expression of ECM molecules and axon growth inhibitors in cortical astrocytes and meningeal fibroblasts cultured separately and in the co-cultures. Levels of each target mRNA were normalized to the housekeeping gene cyclophilin. Statistics: unpaired T-test * p < 0.05, ** p < 0.01. (B) Effects of scar-reducing treatments. Plotted are the levels of target mRNAs normalized to cyclophilin relative to the H2O control treatment. DFO reduced the levels of collagen IV and NG-2. Treatment with cAMP led to a reduction in Tnc. Neurocan and phosphacan were upregulated in DFO + cAMP, whereas NG-2 was significantly reduced after DFO treatment and in DFO + cAMP co-treatments compared to cAMP alone. Statistics: one way Anova with Bonferroni * 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.g004: Effects of TGF-β and scar-reducing treatments on gene expression.(A) Effects of TGF-β on the mRNA expression of ECM molecules and axon growth inhibitors in cortical astrocytes and meningeal fibroblasts cultured separately and in the co-cultures. Levels of each target mRNA were normalized to the housekeeping gene cyclophilin. Statistics: unpaired T-test * p < 0.05, ** p < 0.01. (B) Effects of scar-reducing treatments. Plotted are the levels of target mRNAs normalized to cyclophilin relative to the H2O control treatment. DFO reduced the levels of collagen IV and NG-2. Treatment with cAMP led to a reduction in Tnc. Neurocan and phosphacan were upregulated in DFO + cAMP, whereas NG-2 was significantly reduced after DFO treatment and in DFO + cAMP co-treatments compared to cAMP alone. Statistics: one way Anova with Bonferroni * p < 0.05, ** p < 0.01, *** p< 0.001.
Mentions: We studied the mRNA expression of various ECM molecules and axon growth inhibitors by real time quantitative PCR (Fig 4, Panel A). We extracted RNA from the astrocyte-fibroblast co-cultures as well as from the individual cell mono-cultures, treated with and without TGF-β, in order to investigate the relative contribution of the two cell types. We normalized to cyclophilin, a housekeeping gene that stayed constant after TGF treatment as opposed to GAPDH and ODC which were both regulated by TGF (data not shown). The relative expression levels of the molecules of interest allowed us to estimate their relative contribution to the scar matrix.

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