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Long-term treatment with PP2 after spinal cord injury resulted in functional locomotor recovery and increased spared tissue.

Rosas OR, Torrado AI, Santiago JM, Rodriguez AE, Salgado IK, Miranda JD - Neural Regen Res (2014)

Bottom Line: However, blockade of SFK activity did not change the astrocytic response or infiltration of cells from the immune system at 28 days post-injury.Together, these findings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiology generated after spinal cord injury.Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA.

ABSTRACT
The spinal cord has the ability to regenerate but the microenvironment generated after trauma reduces that capacity. An increase in Src family kinase (SFK) activity has been implicated in neuropathological conditions associated with central nervous system trauma. Therefore, we hypothesized that a decrease in SFK activation by a long-term treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2), a selective SFK inhibitor, after spinal cord contusion with the New York University (NYU) impactor device would generate a permissive environment that improves axonal sprouting and/or behavioral activity. Results demonstrated that long-term blockade of SFK activation with PP2 increases locomotor activity at 7, 14, 21 and 28 days post-injury in the Basso, Beattie, and Bresnahan open field test, round and square beam crossing tests. In addition, an increase in white matter spared tissue and serotonin fiber density was observed in animals treated with PP2. However, blockade of SFK activity did not change the astrocytic response or infiltration of cells from the immune system at 28 days post-injury. Moreover, a reduced SFK activity with PP2 diminished Ephexin (a guanine nucleotide exchange factor) phosphorylation in the acute phase (4 days post-injury) after trauma. Together, these findings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiology generated after spinal cord injury. Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

No MeSH data available.


Related in: MedlinePlus

A blocker of Src family kinases PP2 increased serotonin immunoreactivity caudal to the lesion epicenter after spinal cord injury.(A) Immunoreactive fibers for serotonin (5-hydroxytryptamine) (white arrowheads) were observed caudal (approximately 2 mm) to the lesion epicenter in PP2-treated rats but very few in PP3-treated and DMSO-treated rats. Scale bar: 30 μm. (B) Statistical analysis shows an increase in 5-hydroxytryptamine fiber density caudal to the lesion epicenter in PP2-treated rats (**P < 0.01 for PP3-treated rats and ***P < 0.001 for DMSO-treated rats) (n = 3). One-way analysis of variance followed by Bonferroni post hoc test was used to determine the significant differences among samples studied (F(2,6) = 34.80, P = 0.0005). Error bars show the standard error of the mean (SEM). PP2: 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine; PP3: 4-amino-7-phenylpyrazolo[3,4-d]pyramidine (the inactive analog of PP2); DMSO: dimethyl sulfoxide.
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Figure 3: A blocker of Src family kinases PP2 increased serotonin immunoreactivity caudal to the lesion epicenter after spinal cord injury.(A) Immunoreactive fibers for serotonin (5-hydroxytryptamine) (white arrowheads) were observed caudal (approximately 2 mm) to the lesion epicenter in PP2-treated rats but very few in PP3-treated and DMSO-treated rats. Scale bar: 30 μm. (B) Statistical analysis shows an increase in 5-hydroxytryptamine fiber density caudal to the lesion epicenter in PP2-treated rats (**P < 0.01 for PP3-treated rats and ***P < 0.001 for DMSO-treated rats) (n = 3). One-way analysis of variance followed by Bonferroni post hoc test was used to determine the significant differences among samples studied (F(2,6) = 34.80, P = 0.0005). Error bars show the standard error of the mean (SEM). PP2: 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine; PP3: 4-amino-7-phenylpyrazolo[3,4-d]pyramidine (the inactive analog of PP2); DMSO: dimethyl sulfoxide.

Mentions: Anatomical assays using an antibody against serotonin (5-HT) illustrated the presence of fibers caudal (approximately 2 mm) to the lesion epicenter (Figure 3A) in PP2 group (35.67 ± 0.8819) compared to PP3 (P < 0.01, 13.67 ± 2.333) and DMSO (P < 0.001, 10.33 ± 3.180 (Figure 3B)) groups at 28 days post-injury (n = 3). Control experiments performed with IgG1 or saline, instead of the primary antibody against 5-HT, did not show any immunoreactivity (data not shown). One-way analysis of variance followed by Bonferroni post hoc test was used to determine the significant differences among samples studied (F(2,6) = 34.80, P = 0.0005).


Long-term treatment with PP2 after spinal cord injury resulted in functional locomotor recovery and increased spared tissue.

Rosas OR, Torrado AI, Santiago JM, Rodriguez AE, Salgado IK, Miranda JD - Neural Regen Res (2014)

A blocker of Src family kinases PP2 increased serotonin immunoreactivity caudal to the lesion epicenter after spinal cord injury.(A) Immunoreactive fibers for serotonin (5-hydroxytryptamine) (white arrowheads) were observed caudal (approximately 2 mm) to the lesion epicenter in PP2-treated rats but very few in PP3-treated and DMSO-treated rats. Scale bar: 30 μm. (B) Statistical analysis shows an increase in 5-hydroxytryptamine fiber density caudal to the lesion epicenter in PP2-treated rats (**P < 0.01 for PP3-treated rats and ***P < 0.001 for DMSO-treated rats) (n = 3). One-way analysis of variance followed by Bonferroni post hoc test was used to determine the significant differences among samples studied (F(2,6) = 34.80, P = 0.0005). Error bars show the standard error of the mean (SEM). PP2: 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine; PP3: 4-amino-7-phenylpyrazolo[3,4-d]pyramidine (the inactive analog of PP2); DMSO: dimethyl sulfoxide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4316450&req=5

Figure 3: A blocker of Src family kinases PP2 increased serotonin immunoreactivity caudal to the lesion epicenter after spinal cord injury.(A) Immunoreactive fibers for serotonin (5-hydroxytryptamine) (white arrowheads) were observed caudal (approximately 2 mm) to the lesion epicenter in PP2-treated rats but very few in PP3-treated and DMSO-treated rats. Scale bar: 30 μm. (B) Statistical analysis shows an increase in 5-hydroxytryptamine fiber density caudal to the lesion epicenter in PP2-treated rats (**P < 0.01 for PP3-treated rats and ***P < 0.001 for DMSO-treated rats) (n = 3). One-way analysis of variance followed by Bonferroni post hoc test was used to determine the significant differences among samples studied (F(2,6) = 34.80, P = 0.0005). Error bars show the standard error of the mean (SEM). PP2: 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine; PP3: 4-amino-7-phenylpyrazolo[3,4-d]pyramidine (the inactive analog of PP2); DMSO: dimethyl sulfoxide.
Mentions: Anatomical assays using an antibody against serotonin (5-HT) illustrated the presence of fibers caudal (approximately 2 mm) to the lesion epicenter (Figure 3A) in PP2 group (35.67 ± 0.8819) compared to PP3 (P < 0.01, 13.67 ± 2.333) and DMSO (P < 0.001, 10.33 ± 3.180 (Figure 3B)) groups at 28 days post-injury (n = 3). Control experiments performed with IgG1 or saline, instead of the primary antibody against 5-HT, did not show any immunoreactivity (data not shown). One-way analysis of variance followed by Bonferroni post hoc test was used to determine the significant differences among samples studied (F(2,6) = 34.80, P = 0.0005).

Bottom Line: However, blockade of SFK activity did not change the astrocytic response or infiltration of cells from the immune system at 28 days post-injury.Together, these findings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiology generated after spinal cord injury.Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA.

ABSTRACT
The spinal cord has the ability to regenerate but the microenvironment generated after trauma reduces that capacity. An increase in Src family kinase (SFK) activity has been implicated in neuropathological conditions associated with central nervous system trauma. Therefore, we hypothesized that a decrease in SFK activation by a long-term treatment with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine (PP2), a selective SFK inhibitor, after spinal cord contusion with the New York University (NYU) impactor device would generate a permissive environment that improves axonal sprouting and/or behavioral activity. Results demonstrated that long-term blockade of SFK activation with PP2 increases locomotor activity at 7, 14, 21 and 28 days post-injury in the Basso, Beattie, and Bresnahan open field test, round and square beam crossing tests. In addition, an increase in white matter spared tissue and serotonin fiber density was observed in animals treated with PP2. However, blockade of SFK activity did not change the astrocytic response or infiltration of cells from the immune system at 28 days post-injury. Moreover, a reduced SFK activity with PP2 diminished Ephexin (a guanine nucleotide exchange factor) phosphorylation in the acute phase (4 days post-injury) after trauma. Together, these findings suggest a potential role of SFK in the regulation of spared tissue and/or axonal outgrowth that may result in functional locomotor recovery during the pathophysiology generated after spinal cord injury. Our study also points out that ephexin1 phosphorylation (activation) by SFK action may be involved in the repulsive microenvironment generated after spinal cord injury.

No MeSH data available.


Related in: MedlinePlus