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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

Schematic overview of the in vitro scarring model.Droplets of cortical astrocytes (A) and meningeal fibroblasts (F) were plated and allowed to grow in monolayers that contacted each other between 7 and 14 days. Then, TGF-β1 was added and incubated for 7 d. During this period, clusters were formed by the fibroblasts and those that appeared at the fibroblast to astrocyte border were surrounded by astrocytes. At 7 d after TGF-β-stimulation, dissociated neonatal cortical neurons (in red) were plated and allowed to grow for another 3 d. Potential scar-reducing treatments were applied starting from the time point of TGF stimulation. (a-d) Immunocytochemical staining for fibronectin (red), GFAP (green) and DAPI (blue) showing no cluster formation in astrocyte–fibroblast co-cultures without TGF-β. (e-h) TGF-β induced cluster formation. Clusters formed at the border of the two cell types consisted of meningeal fibroblasts surrounded by astrocytes (arrows). (i-l) Cluster formation was abolished by the TGF-β inhibitor LY364947. Scale bar = 100 μm.
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pone.0134371.g001: Schematic overview of the in vitro scarring model.Droplets of cortical astrocytes (A) and meningeal fibroblasts (F) were plated and allowed to grow in monolayers that contacted each other between 7 and 14 days. Then, TGF-β1 was added and incubated for 7 d. During this period, clusters were formed by the fibroblasts and those that appeared at the fibroblast to astrocyte border were surrounded by astrocytes. At 7 d after TGF-β-stimulation, dissociated neonatal cortical neurons (in red) were plated and allowed to grow for another 3 d. Potential scar-reducing treatments were applied starting from the time point of TGF stimulation. (a-d) Immunocytochemical staining for fibronectin (red), GFAP (green) and DAPI (blue) showing no cluster formation in astrocyte–fibroblast co-cultures without TGF-β. (e-h) TGF-β induced cluster formation. Clusters formed at the border of the two cell types consisted of meningeal fibroblasts surrounded by astrocytes (arrows). (i-l) Cluster formation was abolished by the TGF-β inhibitor LY364947. Scale bar = 100 μm.

Mentions: After propagation of the cortical astrocytes and meningeal fibroblasts (maximally 1 and 5 passages respectively) the cells were deposited as droplets containing 15,000 cells each (Fig 1) onto coverslips coated with 0.1 mg/ml poly-D lysine (Sigma). These were allowed to settle for max. 5 h after which they received GlutaMAX medium containing 10% FBS, 2 mM L-glutamine and 50 Units P/S. The co-cultures were incubated for 10–16 d in a 37°C incubator under 10% CO2 until the layers were confluent and contacted each other. Subsequently, 10 ng/ml of recombinant human TGF-β1 (R&D Systems) was added to the medium and incubated for 7 d, inducing scar-like cluster formation. At 7 d after the treatment of the co-cultures with TGF-β1 50,000 cortical neurons per well were added to the culture medium and incubated for 3 more days. Medium was refreshed 1 d prior to plating of the neurons, so that the astrocytes had sufficient time to condition the medium for axon growth.


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)

Schematic overview of the in vitro scarring model.Droplets of cortical astrocytes (A) and meningeal fibroblasts (F) were plated and allowed to grow in monolayers that contacted each other between 7 and 14 days. Then, TGF-β1 was added and incubated for 7 d. During this period, clusters were formed by the fibroblasts and those that appeared at the fibroblast to astrocyte border were surrounded by astrocytes. At 7 d after TGF-β-stimulation, dissociated neonatal cortical neurons (in red) were plated and allowed to grow for another 3 d. Potential scar-reducing treatments were applied starting from the time point of TGF stimulation. (a-d) Immunocytochemical staining for fibronectin (red), GFAP (green) and DAPI (blue) showing no cluster formation in astrocyte–fibroblast co-cultures without TGF-β. (e-h) TGF-β induced cluster formation. Clusters formed at the border of the two cell types consisted of meningeal fibroblasts surrounded by astrocytes (arrows). (i-l) Cluster formation was abolished by the TGF-β inhibitor LY364947. Scale bar = 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134371.g001: Schematic overview of the in vitro scarring model.Droplets of cortical astrocytes (A) and meningeal fibroblasts (F) were plated and allowed to grow in monolayers that contacted each other between 7 and 14 days. Then, TGF-β1 was added and incubated for 7 d. During this period, clusters were formed by the fibroblasts and those that appeared at the fibroblast to astrocyte border were surrounded by astrocytes. At 7 d after TGF-β-stimulation, dissociated neonatal cortical neurons (in red) were plated and allowed to grow for another 3 d. Potential scar-reducing treatments were applied starting from the time point of TGF stimulation. (a-d) Immunocytochemical staining for fibronectin (red), GFAP (green) and DAPI (blue) showing no cluster formation in astrocyte–fibroblast co-cultures without TGF-β. (e-h) TGF-β induced cluster formation. Clusters formed at the border of the two cell types consisted of meningeal fibroblasts surrounded by astrocytes (arrows). (i-l) Cluster formation was abolished by the TGF-β inhibitor LY364947. Scale bar = 100 μm.
Mentions: After propagation of the cortical astrocytes and meningeal fibroblasts (maximally 1 and 5 passages respectively) the cells were deposited as droplets containing 15,000 cells each (Fig 1) onto coverslips coated with 0.1 mg/ml poly-D lysine (Sigma). These were allowed to settle for max. 5 h after which they received GlutaMAX medium containing 10% FBS, 2 mM L-glutamine and 50 Units P/S. The co-cultures were incubated for 10–16 d in a 37°C incubator under 10% CO2 until the layers were confluent and contacted each other. Subsequently, 10 ng/ml of recombinant human TGF-β1 (R&D Systems) was added to the medium and incubated for 7 d, inducing scar-like cluster formation. At 7 d after the treatment of the co-cultures with TGF-β1 50,000 cortical neurons per well were added to the culture medium and incubated for 3 more days. Medium was refreshed 1 d prior to plating of the neurons, so that the astrocytes had sufficient time to condition the medium for axon growth.

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