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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

Reduction of scar density measured by the quantification of ECM Coll IV.Mosaic composite pictures of the complete scar in spinal cord injured animals after treatment with (A, B) PBS corresponding to (C, D) DFO treatment, (E, F) PBS corresponding to (G, H) cAMP treatment and (I, J) Tris corresponding to (K, L) BPY-DCA treatment. The tissue was stained for Coll IV in green (A, C, E, G, I, K) and vWF in red (B, D, F, H, J, L). Quantification of ECM Coll IV was performed by subtraction of vWF-positive blood vessels. The remaining signals for ECM Coll IV for the respective treatments were plotted in M, N and O. A significant reduction of Coll IV was observed in both treatment concentrations for BPY-DCA and DFO as well as in the highest concentration of cAMP. High magnification pictures of the Coll IV staining in part of the lesion site are provided in S2 Fig. Statistics: one way Anova with Dunnett’s post-hoc test * p < 0.05, ** p < 0.01, *** p< 0.001 Scale bar (in A) = 200 μm, applies for all pictures.
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pone.0134371.g008: Reduction of scar density measured by the quantification of ECM Coll IV.Mosaic composite pictures of the complete scar in spinal cord injured animals after treatment with (A, B) PBS corresponding to (C, D) DFO treatment, (E, F) PBS corresponding to (G, H) cAMP treatment and (I, J) Tris corresponding to (K, L) BPY-DCA treatment. The tissue was stained for Coll IV in green (A, C, E, G, I, K) and vWF in red (B, D, F, H, J, L). Quantification of ECM Coll IV was performed by subtraction of vWF-positive blood vessels. The remaining signals for ECM Coll IV for the respective treatments were plotted in M, N and O. A significant reduction of Coll IV was observed in both treatment concentrations for BPY-DCA and DFO as well as in the highest concentration of cAMP. High magnification pictures of the Coll IV staining in part of the lesion site are provided in S2 Fig. Statistics: one way Anova with Dunnett’s post-hoc test * p < 0.05, ** p < 0.01, *** p< 0.001 Scale bar (in A) = 200 μm, applies for all pictures.

Mentions: The in vitro results suggest that DFO treatment may be preferable over the existing AST strategy with respect to reduction in number and size of scar-like clusters as well as neurite length on clusters. The combination of iron chelators with cAMP reduced the number of clusters slightly more, but also led to undesirable upregulation of phosphacan and neurocan mRNA. To prove the efficacy of DFO in vivo we investigated the scar-reducing capabilities of DFO in comparison to BPY-DCA and cAMP in the dorsal hemisection spinal cord injury model in rats. The treatments were applied by local intrathecal infusion for 1 week using osmotic minipumps. We applied 10 and 50 μg DFO per day, which would correspond to roughly 30 and 150 μM assuming a CSF volume of 500 μl in rat [57]. For BPY-DCA, the 1.1 or 7.8 μg/d would be about 9 or 70 μM. The amount of cAMP applied was 50 and 100 μg/d (0.2 and 0.4 mM). These calculations fall roughly within the range of the concentrations used in the in vitro model. As a measure for scar density we then semi-quantitatively determined the amount of Coll IV in the ECM of the scar by immunohistochemistry [8]. For this purpose, we double stained with an antibody directed to vWF to correct for the amount of blood vessels in the scar (Fig 8). Since Coll IV is also expressed in endothelial cells [58, 59] and iron chelators, like DFO, are able to promote angiogenesis ([60, 61]; and own observations), the Coll IV-stained blood vessels in the scar will confuse the quantification of the ECM Coll IV. A significant decrease in ECM Coll IV immunoreactivity was observed for BPY-DCA and DFO at all concentrations applied (Fig 8M and 8O). In relation to their respective controls, BPY-DCA led to an ECM collagen IV reduction of about 36%, while DFO showed a reducing effect of approximately 30%. The application of 100 μg/d cAMP also reduced Coll IV significantly in comparison to its control (Fig 8N). Application of 10 μg/d DFO for 2 weeks resulted in a similar extent of scar-reduction (data not shown).


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)

Reduction of scar density measured by the quantification of ECM Coll IV.Mosaic composite pictures of the complete scar in spinal cord injured animals after treatment with (A, B) PBS corresponding to (C, D) DFO treatment, (E, F) PBS corresponding to (G, H) cAMP treatment and (I, J) Tris corresponding to (K, L) BPY-DCA treatment. The tissue was stained for Coll IV in green (A, C, E, G, I, K) and vWF in red (B, D, F, H, J, L). Quantification of ECM Coll IV was performed by subtraction of vWF-positive blood vessels. The remaining signals for ECM Coll IV for the respective treatments were plotted in M, N and O. A significant reduction of Coll IV was observed in both treatment concentrations for BPY-DCA and DFO as well as in the highest concentration of cAMP. High magnification pictures of the Coll IV staining in part of the lesion site are provided in S2 Fig. Statistics: one way Anova with Dunnett’s post-hoc test * p < 0.05, ** p < 0.01, *** p< 0.001 Scale bar (in A) = 200 μm, applies for all pictures.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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pone.0134371.g008: Reduction of scar density measured by the quantification of ECM Coll IV.Mosaic composite pictures of the complete scar in spinal cord injured animals after treatment with (A, B) PBS corresponding to (C, D) DFO treatment, (E, F) PBS corresponding to (G, H) cAMP treatment and (I, J) Tris corresponding to (K, L) BPY-DCA treatment. The tissue was stained for Coll IV in green (A, C, E, G, I, K) and vWF in red (B, D, F, H, J, L). Quantification of ECM Coll IV was performed by subtraction of vWF-positive blood vessels. The remaining signals for ECM Coll IV for the respective treatments were plotted in M, N and O. A significant reduction of Coll IV was observed in both treatment concentrations for BPY-DCA and DFO as well as in the highest concentration of cAMP. High magnification pictures of the Coll IV staining in part of the lesion site are provided in S2 Fig. Statistics: one way Anova with Dunnett’s post-hoc test * p < 0.05, ** p < 0.01, *** p< 0.001 Scale bar (in A) = 200 μm, applies for all pictures.
Mentions: The in vitro results suggest that DFO treatment may be preferable over the existing AST strategy with respect to reduction in number and size of scar-like clusters as well as neurite length on clusters. The combination of iron chelators with cAMP reduced the number of clusters slightly more, but also led to undesirable upregulation of phosphacan and neurocan mRNA. To prove the efficacy of DFO in vivo we investigated the scar-reducing capabilities of DFO in comparison to BPY-DCA and cAMP in the dorsal hemisection spinal cord injury model in rats. The treatments were applied by local intrathecal infusion for 1 week using osmotic minipumps. We applied 10 and 50 μg DFO per day, which would correspond to roughly 30 and 150 μM assuming a CSF volume of 500 μl in rat [57]. For BPY-DCA, the 1.1 or 7.8 μg/d would be about 9 or 70 μM. The amount of cAMP applied was 50 and 100 μg/d (0.2 and 0.4 mM). These calculations fall roughly within the range of the concentrations used in the in vitro model. As a measure for scar density we then semi-quantitatively determined the amount of Coll IV in the ECM of the scar by immunohistochemistry [8]. For this purpose, we double stained with an antibody directed to vWF to correct for the amount of blood vessels in the scar (Fig 8). Since Coll IV is also expressed in endothelial cells [58, 59] and iron chelators, like DFO, are able to promote angiogenesis ([60, 61]; and own observations), the Coll IV-stained blood vessels in the scar will confuse the quantification of the ECM Coll IV. A significant decrease in ECM Coll IV immunoreactivity was observed for BPY-DCA and DFO at all concentrations applied (Fig 8M and 8O). In relation to their respective controls, BPY-DCA led to an ECM collagen IV reduction of about 36%, while DFO showed a reducing effect of approximately 30%. The application of 100 μg/d cAMP also reduced Coll IV significantly in comparison to its control (Fig 8N). Application of 10 μg/d DFO for 2 weeks resulted in a similar extent of scar-reduction (data not shown).

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