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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 scarring and regeneration.Tissue preservation, lesion size and axon regeneration at 19 dpl. (A) Exemplary mosaic compositions of PBS- (A) and DFO- (B) treated spinal cord stained with anti-GFAP to visualize the GFAP-negative scar area. Lesion area marked with dotted lines, Asterisks indicate a cystic area in both treatment groups. S3 Fig provides pictures A and B with higher intensity to show that the lesion site is filled with tissue. Straight lines outline the region of interest used for quantification of lesion site (2.5 mm). DFO animals had significantly smaller lesion areas (corrected for the total area) than PBS controls (C) and significantly more tissue sparing (D) and. (E) Quantification of the number of CST axons per mm2 of lesion area revealed a significant increase. (F) the CST tracing was equal in both groups. (G) The number of CGRP axons in the scar was significantly increased in DFO-treated rats. (H, I) Representative pictures of CGRP axon profiles (arrows) in the lesion area in DFO- and PBS-treated animals. Note that the red spots in I originate from aberrantly stained macrophages. Statistics: unpaired T-test * p < 0.05, ** p < 0.01. Scale bar (G, H) = 50 μm.
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pone.0134371.g010: Effects of DFO on scarring and regeneration.Tissue preservation, lesion size and axon regeneration at 19 dpl. (A) Exemplary mosaic compositions of PBS- (A) and DFO- (B) treated spinal cord stained with anti-GFAP to visualize the GFAP-negative scar area. Lesion area marked with dotted lines, Asterisks indicate a cystic area in both treatment groups. S3 Fig provides pictures A and B with higher intensity to show that the lesion site is filled with tissue. Straight lines outline the region of interest used for quantification of lesion site (2.5 mm). DFO animals had significantly smaller lesion areas (corrected for the total area) than PBS controls (C) and significantly more tissue sparing (D) and. (E) Quantification of the number of CST axons per mm2 of lesion area revealed a significant increase. (F) the CST tracing was equal in both groups. (G) The number of CGRP axons in the scar was significantly increased in DFO-treated rats. (H, I) Representative pictures of CGRP axon profiles (arrows) in the lesion area in DFO- and PBS-treated animals. Note that the red spots in I originate from aberrantly stained macrophages. Statistics: unpaired T-test * p < 0.05, ** p < 0.01. Scale bar (G, H) = 50 μm.

Mentions: At 19 weeks post-lesion, the animals were perfused and the spinal cords analyzed. By quantification of the GFAP-negative scar area, we found a significant reduction of the lesion size by DFO as compared to the PBS control (Fig 10A and 10C). Moreover, we observed that DFO-treated spinal cords in general were thicker around the lesion zone than PBS controls. Quantification of a 2.5 mm stretch of tissue around the lesion site (1.25 mm in each direction measured from the lesion centre), in sections where the central channel was included, revealed a DFO-induced increase in spared tissue (Fig 10D). Axon regeneration was studied in the same animals by counting BDA-traced descending CST axons and CGRP-stained ascending axons. A significant increase in the number of CGRP-positive axons profiles per mm2 of scar area was observed in DFO-treated animals as compared to controls (Fig 10G–10I). A small number of regenerating CST axons was found in the scar area, which was significantly increased in the DFO-treated animals (Fig 10E). There was no difference in the efficiency of CST tracing between the groups (Fig 10F).


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 scarring and regeneration.Tissue preservation, lesion size and axon regeneration at 19 dpl. (A) Exemplary mosaic compositions of PBS- (A) and DFO- (B) treated spinal cord stained with anti-GFAP to visualize the GFAP-negative scar area. Lesion area marked with dotted lines, Asterisks indicate a cystic area in both treatment groups. S3 Fig provides pictures A and B with higher intensity to show that the lesion site is filled with tissue. Straight lines outline the region of interest used for quantification of lesion site (2.5 mm). DFO animals had significantly smaller lesion areas (corrected for the total area) than PBS controls (C) and significantly more tissue sparing (D) and. (E) Quantification of the number of CST axons per mm2 of lesion area revealed a significant increase. (F) the CST tracing was equal in both groups. (G) The number of CGRP axons in the scar was significantly increased in DFO-treated rats. (H, I) Representative pictures of CGRP axon profiles (arrows) in the lesion area in DFO- and PBS-treated animals. Note that the red spots in I originate from aberrantly stained macrophages. Statistics: unpaired T-test * p < 0.05, ** p < 0.01. Scale bar (G, H) = 50 μm.
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pone.0134371.g010: Effects of DFO on scarring and regeneration.Tissue preservation, lesion size and axon regeneration at 19 dpl. (A) Exemplary mosaic compositions of PBS- (A) and DFO- (B) treated spinal cord stained with anti-GFAP to visualize the GFAP-negative scar area. Lesion area marked with dotted lines, Asterisks indicate a cystic area in both treatment groups. S3 Fig provides pictures A and B with higher intensity to show that the lesion site is filled with tissue. Straight lines outline the region of interest used for quantification of lesion site (2.5 mm). DFO animals had significantly smaller lesion areas (corrected for the total area) than PBS controls (C) and significantly more tissue sparing (D) and. (E) Quantification of the number of CST axons per mm2 of lesion area revealed a significant increase. (F) the CST tracing was equal in both groups. (G) The number of CGRP axons in the scar was significantly increased in DFO-treated rats. (H, I) Representative pictures of CGRP axon profiles (arrows) in the lesion area in DFO- and PBS-treated animals. Note that the red spots in I originate from aberrantly stained macrophages. Statistics: unpaired T-test * p < 0.05, ** p < 0.01. Scale bar (G, H) = 50 μm.
Mentions: At 19 weeks post-lesion, the animals were perfused and the spinal cords analyzed. By quantification of the GFAP-negative scar area, we found a significant reduction of the lesion size by DFO as compared to the PBS control (Fig 10A and 10C). Moreover, we observed that DFO-treated spinal cords in general were thicker around the lesion zone than PBS controls. Quantification of a 2.5 mm stretch of tissue around the lesion site (1.25 mm in each direction measured from the lesion centre), in sections where the central channel was included, revealed a DFO-induced increase in spared tissue (Fig 10D). Axon regeneration was studied in the same animals by counting BDA-traced descending CST axons and CGRP-stained ascending axons. A significant increase in the number of CGRP-positive axons profiles per mm2 of scar area was observed in DFO-treated animals as compared to controls (Fig 10G–10I). A small number of regenerating CST axons was found in the scar area, which was significantly increased in the DFO-treated animals (Fig 10E). There was no difference in the efficiency of CST tracing between the groups (Fig 10F).

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