Limits...
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

Cluster composition, correlation with neurite growth.(A) ICC for selected ECM and axon growth inhibitory molecules stained with infrared-labelled secondary antibodies in the 7-days-old clusters. Scale bar = 100 μm. (B) Co-culture with neonatal cortical neurons. ICC staining with GFAP, CS-56 (a general marker for CSPGs), NG-2 and Tnc in infrared, β-III-Tubulin in cyan, DAPI in blue. Neurites tended to co-localize with CSPG-positive astrocytes (arrow). Neurites were short and avoided the cluster center. Tnc was expressed by the neurons (arrowheads) as well as by the fibroblasts. Scale bar = 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4519270&req=5

pone.0134371.g003: Cluster composition, correlation with neurite growth.(A) ICC for selected ECM and axon growth inhibitory molecules stained with infrared-labelled secondary antibodies in the 7-days-old clusters. Scale bar = 100 μm. (B) Co-culture with neonatal cortical neurons. ICC staining with GFAP, CS-56 (a general marker for CSPGs), NG-2 and Tnc in infrared, β-III-Tubulin in cyan, DAPI in blue. Neurites tended to co-localize with CSPG-positive astrocytes (arrow). Neurites were short and avoided the cluster center. Tnc was expressed by the neurons (arrowheads) as well as by the fibroblasts. Scale bar = 50 μm.

Mentions: To confirm that the composition of the clusters resembled the lesion scar in SCI, we performed immunocytochemistry (ICC) for several ECM molecules and axon growth inhibitors known to accumulate in the fibrous scar in vivo. Due to high autofluorescence in the green wavelength and some autofluorescence in visible red, all scar components were visualized with infrared dyes (Alexa 647). We observed immunoreactivity for Coll IV, Tnc, and Sem3A as well as for the CSPGs NG-2, neurocan and phosphacan (Fig 3A and 3B). Ephrin B2 and EphB2 were expressed at very low levels (data not shown) and controls lacking primary antibodies were negative (Fig 3A last panels). For most molecules, higher levels of immunoreactivity were observed at the border and surface of the clusters compared to the center (Fig 3A). Dissociated neonatal cortical neurons grew βIII-Tubulin-positive neurites on both fibroblast and astroglial cell layers. Neurite growth was less pronounced on top of the clusters and often confined to areas where astrocytes were present (Fig 3B). Neurites mostly did not cross the border between the monolayer and the cluster, where often high concentrations of growth inhibitors were found (Fig 3A and 3B).


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)

Cluster composition, correlation with neurite growth.(A) ICC for selected ECM and axon growth inhibitory molecules stained with infrared-labelled secondary antibodies in the 7-days-old clusters. Scale bar = 100 μm. (B) Co-culture with neonatal cortical neurons. ICC staining with GFAP, CS-56 (a general marker for CSPGs), NG-2 and Tnc in infrared, β-III-Tubulin in cyan, DAPI in blue. Neurites tended to co-localize with CSPG-positive astrocytes (arrow). Neurites were short and avoided the cluster center. Tnc was expressed by the neurons (arrowheads) as well as by the fibroblasts. Scale bar = 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134371.g003: Cluster composition, correlation with neurite growth.(A) ICC for selected ECM and axon growth inhibitory molecules stained with infrared-labelled secondary antibodies in the 7-days-old clusters. Scale bar = 100 μm. (B) Co-culture with neonatal cortical neurons. ICC staining with GFAP, CS-56 (a general marker for CSPGs), NG-2 and Tnc in infrared, β-III-Tubulin in cyan, DAPI in blue. Neurites tended to co-localize with CSPG-positive astrocytes (arrow). Neurites were short and avoided the cluster center. Tnc was expressed by the neurons (arrowheads) as well as by the fibroblasts. Scale bar = 50 μm.
Mentions: To confirm that the composition of the clusters resembled the lesion scar in SCI, we performed immunocytochemistry (ICC) for several ECM molecules and axon growth inhibitors known to accumulate in the fibrous scar in vivo. Due to high autofluorescence in the green wavelength and some autofluorescence in visible red, all scar components were visualized with infrared dyes (Alexa 647). We observed immunoreactivity for Coll IV, Tnc, and Sem3A as well as for the CSPGs NG-2, neurocan and phosphacan (Fig 3A and 3B). Ephrin B2 and EphB2 were expressed at very low levels (data not shown) and controls lacking primary antibodies were negative (Fig 3A last panels). For most molecules, higher levels of immunoreactivity were observed at the border and surface of the clusters compared to the center (Fig 3A). Dissociated neonatal cortical neurons grew βIII-Tubulin-positive neurites on both fibroblast and astroglial cell layers. Neurite growth was less pronounced on top of the clusters and often confined to areas where astrocytes were present (Fig 3B). Neurites mostly did not cross the border between the monolayer and the cluster, where often high concentrations of growth inhibitors were found (Fig 3A and 3B).

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