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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 DFO on locomotor recovery.Long-term locomotor behavior in DFO-treated animals was assessed using the (A) BBB score and subscore, (B) Gridwalk and (C) Catwalk analysis. Left and right hindpaws were analyzed separately, because of asymmetry of the lesion, with the left rubrospinal tract being more affected than the right. Both BBB subscore and the Catwalk showed a positive but non-significant trend for DFO as compared to PBS controls. On the Gridwalk DFO-treated rats performed better than PBS controls. The percentage of mistakes of the right hindlimb decreased significantly during 2–10 weeks after injury, but was not significantly different from PBS controls during the last 4 weeks. Statistics: Mann-Whitney test * p < 0.05, ** p < 0.01, *** p< 0.001.
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pone.0134371.g009: Effects of DFO on locomotor recovery.Long-term locomotor behavior in DFO-treated animals was assessed using the (A) BBB score and subscore, (B) Gridwalk and (C) Catwalk analysis. Left and right hindpaws were analyzed separately, because of asymmetry of the lesion, with the left rubrospinal tract being more affected than the right. Both BBB subscore and the Catwalk showed a positive but non-significant trend for DFO as compared to PBS controls. On the Gridwalk DFO-treated rats performed better than PBS controls. The percentage of mistakes of the right hindlimb decreased significantly during 2–10 weeks after injury, but was not significantly different from PBS controls during the last 4 weeks. Statistics: Mann-Whitney test * p < 0.05, ** p < 0.01, *** p< 0.001.

Mentions: We applied 10 μg/d DFO for 2 weeks for a long-term effects behavioral study of 19 weeks. Locomotor recovery was evaluated over 1, 2, 12 and 16 weeks post-lesion using the open field locomotor BBB score and subscore. The horizontal ladder test (Gridwalk) and the Catwalk automated gait analysis were performed every second week. We evaluated the hindlimbs separately, because of a slight asymmetry in our Scouten wire knife lesion, where the right rubrospinal tract is less impaired than the left rubrospinal tract [10]. The BBB subscore showed a positive but non-significant trend for both paws in DFO-treated animals (Fig 9A). During the Gridwalk analysis the DFO-treated animals showed significantly less foot slips with the right hindlimb than the controls at 2, 6, 8 and 10 weeks. However, the later timepoints were not significantly different from the PBS control group (Fig 9B). A positive trend, but no significant difference was observed at 4 and 6 weeks for the left hindlimb. The Catwalk analysis revealed a positive but non-significant trend for DFO in the regularity index, a measure for limb coordination (Fig 9C). Similar to the Gridwalk, this trend disappeared at later timepoints.


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 DFO on locomotor recovery.Long-term locomotor behavior in DFO-treated animals was assessed using the (A) BBB score and subscore, (B) Gridwalk and (C) Catwalk analysis. Left and right hindpaws were analyzed separately, because of asymmetry of the lesion, with the left rubrospinal tract being more affected than the right. Both BBB subscore and the Catwalk showed a positive but non-significant trend for DFO as compared to PBS controls. On the Gridwalk DFO-treated rats performed better than PBS controls. The percentage of mistakes of the right hindlimb decreased significantly during 2–10 weeks after injury, but was not significantly different from PBS controls during the last 4 weeks. Statistics: Mann-Whitney 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.g009: Effects of DFO on locomotor recovery.Long-term locomotor behavior in DFO-treated animals was assessed using the (A) BBB score and subscore, (B) Gridwalk and (C) Catwalk analysis. Left and right hindpaws were analyzed separately, because of asymmetry of the lesion, with the left rubrospinal tract being more affected than the right. Both BBB subscore and the Catwalk showed a positive but non-significant trend for DFO as compared to PBS controls. On the Gridwalk DFO-treated rats performed better than PBS controls. The percentage of mistakes of the right hindlimb decreased significantly during 2–10 weeks after injury, but was not significantly different from PBS controls during the last 4 weeks. Statistics: Mann-Whitney test * p < 0.05, ** p < 0.01, *** p< 0.001.
Mentions: We applied 10 μg/d DFO for 2 weeks for a long-term effects behavioral study of 19 weeks. Locomotor recovery was evaluated over 1, 2, 12 and 16 weeks post-lesion using the open field locomotor BBB score and subscore. The horizontal ladder test (Gridwalk) and the Catwalk automated gait analysis were performed every second week. We evaluated the hindlimbs separately, because of a slight asymmetry in our Scouten wire knife lesion, where the right rubrospinal tract is less impaired than the left rubrospinal tract [10]. The BBB subscore showed a positive but non-significant trend for both paws in DFO-treated animals (Fig 9A). During the Gridwalk analysis the DFO-treated animals showed significantly less foot slips with the right hindlimb than the controls at 2, 6, 8 and 10 weeks. However, the later timepoints were not significantly different from the PBS control group (Fig 9B). A positive trend, but no significant difference was observed at 4 and 6 weeks for the left hindlimb. The Catwalk analysis revealed a positive but non-significant trend for DFO in the regularity index, a measure for limb coordination (Fig 9C). Similar to the Gridwalk, this trend disappeared at later timepoints.

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