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Reduction in antioxidant enzyme expression and sustained inflammation enhance tissue damage in the subacute phase of spinal cord contusive injury.

Wang CY, Chen JK, Wu YT, Tsai MJ, Shyue SK, Yang CS, Tzeng SF - J. Biomed. Sci. (2011)

Bottom Line: Our results showed a decline in catalase (CAT) and Mn-superoxide dismutase (MnSOD) found at day 14 after SCI.Delayed treatment with chondroitinase ABC (chABC) at day 3 post SCI improved the hindlimb locomotion in SCI rats.Our findings demonstrate that the differential expression in proteins related to signal transduction, oxidoreduction and stress contribute to extensive inflammation, causing time-dependent spread of tissue damage after severe SCI.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT

Background: Traumatic spinal cord injury (SCI) forms a disadvantageous microenvironment for tissue repair at the lesion site. To consider an appropriate time window for giving a promising therapeutic treatment for subacute and chronic SCI, global changes of proteins in the injured center at the longer survival time points after SCI remains to be elucidated.

Methods: Through two-dimensional electrophoresis (2DE)-based proteome analysis and western blotting, we examined the differential expression of the soluble proteins isolated from the lesion center (LC) at day 1 (acute) and day 14 (subacute) after a severe contusive injury to the thoracic spinal cord at segment 10. In situ apoptotic analysis was used to examine cell apoptosis in injured spinal cord after adenoviral gene transfer of antioxidant enzymes. In addition, administration of chondroitinase ABC (chABC) was performed to analyze hindlimb locomotor recovery in rats with SCI using Basso, Beattie and Bresnahan (BBB) locomotor rating scale.

Results: Our results showed a decline in catalase (CAT) and Mn-superoxide dismutase (MnSOD) found at day 14 after SCI. Accordingly, gene transfer of SOD was introduced in the injured spinal cord and found to attenuate cell apoptosis. Galectin-3, β-actin, actin regulatory protein (CAPG), and F-actin-capping protein subunit β (CAPZB) at day 14 were increased when compared to that detected at day 1 after SCI or in sham-operated control. Indeed, the accumulation of β-actin+ immune cells was observed in the LC at day 14 post SCI, while most of reactive astrocytes were surrounding the lesion center. In addition, chondroitin sulfate proteoglycans (CSPG)-related proteins with 40-kDa was detected in the LC at day 3-14 post SCI. Delayed treatment with chondroitinase ABC (chABC) at day 3 post SCI improved the hindlimb locomotion in SCI rats.

Conclusions: Our findings demonstrate that the differential expression in proteins related to signal transduction, oxidoreduction and stress contribute to extensive inflammation, causing time-dependent spread of tissue damage after severe SCI. The interventions by supplement of anti-oxidant enzymes right after SCI or delayed administration with chABC can facilitate spinal neural cell survival and tissue repair.

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Immunofluorescence for β-actin expression in injured spinal cord tissue sections. The injured spinal cord tissue sections were collected at day 1 (A,B), day 7(C,D), and day 14(E,F) after SCI, and then subjected to immunofluorescence for β-actin (red). The tissues were also subjected for nuclear staining using DAPI (blue). In general, cell debris with β-actin immunoreactivity (open arrowheads) and fragmented/damaged nuclei (inset in A) were exclusively detected in the lesion center (LC) at 1 day post SCI. However, numerous β-actin+ cells (arrows) were accumulated in the LC at day 7 and 14 post SCI. Noted that β-actin+ cells (open arrows) were detected in the distal area (white matter) to the LC at day 1, 7 and 14 post SCI. Scale bar in A-F, 50 μm. (G) Primary astrocytes and microglia prepared from neonatal rat cortical tissues were treated for 24 hours with proinflammatory factor, TNF-α and IL-1β (T/I) at the doses of 20 ng/ml. The total proteins were extracted, separated by SDS-PAGE, and analyzed by western blotting with anti-β-actin, anti-GFAP.
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Figure 4: Immunofluorescence for β-actin expression in injured spinal cord tissue sections. The injured spinal cord tissue sections were collected at day 1 (A,B), day 7(C,D), and day 14(E,F) after SCI, and then subjected to immunofluorescence for β-actin (red). The tissues were also subjected for nuclear staining using DAPI (blue). In general, cell debris with β-actin immunoreactivity (open arrowheads) and fragmented/damaged nuclei (inset in A) were exclusively detected in the lesion center (LC) at 1 day post SCI. However, numerous β-actin+ cells (arrows) were accumulated in the LC at day 7 and 14 post SCI. Noted that β-actin+ cells (open arrows) were detected in the distal area (white matter) to the LC at day 1, 7 and 14 post SCI. Scale bar in A-F, 50 μm. (G) Primary astrocytes and microglia prepared from neonatal rat cortical tissues were treated for 24 hours with proinflammatory factor, TNF-α and IL-1β (T/I) at the doses of 20 ng/ml. The total proteins were extracted, separated by SDS-PAGE, and analyzed by western blotting with anti-β-actin, anti-GFAP.

Mentions: We noticed that β-actin (spot 33) and β-tubulin 5 (spot 22) was biostatistically increased in the LC at day 14, when compared to that detected at day 1 (Figure 1B and Table 2). The intensity of actin filament capping proteins, CAPG (spot 35) and CAPZB (spot 52), were also found increased in 2-DE (Table 2). Western blot analysis also verified that β-actin, CAPG and CAPZB were dramatically increased in the LC at day 14 post SCI (Figure 3). Immunofluorescence also confirmed that β-actin+ cells with an irregular morphology accumulated exclusively in the LC at day 7 and 14 post SCI (Figure 4C, E), while β-actin+ cell debris was detected in the LC at day 1 post SCI (Figure 4A). DAPI nuclei staining indicated that extensive cell death was observed at day 1 post SCI (Figure 4A). We also noticed that β-actin+ cells with a hypertrophic morphology were found at day 1 post SCI in the white matter of the spinal cord distal to the LC(Figure 4B), whereas ramified β-actin+ cells were observed at day 7 and day 14 post SCI (Figure 4D, F). Through proteomic approach, we found that the regulators of inflammation and carboxypeptidase inhibitor, galectin-3 (LEG3; spot 58,59) and latexin (LXN; spot 54), were increased in the LC at day 14 post SCI (Table 2). By Western blot analysis, an increase in LEG3, but not LXN, was found along the longer survival time points (Figure 3). Cathepsin D (CATD), one of lysosomal enzymes enriched in macrophages, was also increased in the LC at day 14 post SCI (Table 2 and Figure 3). Furthermore, immuofluorescence indicated that Iba-1+ microglia were accumulated in the proximal site to the LC (Figure 5A). Moreover, CD11b (Mac-1) or CD49f-positive macrophages were observed in the LC (Figure 5B, C) and the proximal area to the LC at day 14 post SCI (data not shown).


Reduction in antioxidant enzyme expression and sustained inflammation enhance tissue damage in the subacute phase of spinal cord contusive injury.

Wang CY, Chen JK, Wu YT, Tsai MJ, Shyue SK, Yang CS, Tzeng SF - J. Biomed. Sci. (2011)

Immunofluorescence for β-actin expression in injured spinal cord tissue sections. The injured spinal cord tissue sections were collected at day 1 (A,B), day 7(C,D), and day 14(E,F) after SCI, and then subjected to immunofluorescence for β-actin (red). The tissues were also subjected for nuclear staining using DAPI (blue). In general, cell debris with β-actin immunoreactivity (open arrowheads) and fragmented/damaged nuclei (inset in A) were exclusively detected in the lesion center (LC) at 1 day post SCI. However, numerous β-actin+ cells (arrows) were accumulated in the LC at day 7 and 14 post SCI. Noted that β-actin+ cells (open arrows) were detected in the distal area (white matter) to the LC at day 1, 7 and 14 post SCI. Scale bar in A-F, 50 μm. (G) Primary astrocytes and microglia prepared from neonatal rat cortical tissues were treated for 24 hours with proinflammatory factor, TNF-α and IL-1β (T/I) at the doses of 20 ng/ml. The total proteins were extracted, separated by SDS-PAGE, and analyzed by western blotting with anti-β-actin, anti-GFAP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Immunofluorescence for β-actin expression in injured spinal cord tissue sections. The injured spinal cord tissue sections were collected at day 1 (A,B), day 7(C,D), and day 14(E,F) after SCI, and then subjected to immunofluorescence for β-actin (red). The tissues were also subjected for nuclear staining using DAPI (blue). In general, cell debris with β-actin immunoreactivity (open arrowheads) and fragmented/damaged nuclei (inset in A) were exclusively detected in the lesion center (LC) at 1 day post SCI. However, numerous β-actin+ cells (arrows) were accumulated in the LC at day 7 and 14 post SCI. Noted that β-actin+ cells (open arrows) were detected in the distal area (white matter) to the LC at day 1, 7 and 14 post SCI. Scale bar in A-F, 50 μm. (G) Primary astrocytes and microglia prepared from neonatal rat cortical tissues were treated for 24 hours with proinflammatory factor, TNF-α and IL-1β (T/I) at the doses of 20 ng/ml. The total proteins were extracted, separated by SDS-PAGE, and analyzed by western blotting with anti-β-actin, anti-GFAP.
Mentions: We noticed that β-actin (spot 33) and β-tubulin 5 (spot 22) was biostatistically increased in the LC at day 14, when compared to that detected at day 1 (Figure 1B and Table 2). The intensity of actin filament capping proteins, CAPG (spot 35) and CAPZB (spot 52), were also found increased in 2-DE (Table 2). Western blot analysis also verified that β-actin, CAPG and CAPZB were dramatically increased in the LC at day 14 post SCI (Figure 3). Immunofluorescence also confirmed that β-actin+ cells with an irregular morphology accumulated exclusively in the LC at day 7 and 14 post SCI (Figure 4C, E), while β-actin+ cell debris was detected in the LC at day 1 post SCI (Figure 4A). DAPI nuclei staining indicated that extensive cell death was observed at day 1 post SCI (Figure 4A). We also noticed that β-actin+ cells with a hypertrophic morphology were found at day 1 post SCI in the white matter of the spinal cord distal to the LC(Figure 4B), whereas ramified β-actin+ cells were observed at day 7 and day 14 post SCI (Figure 4D, F). Through proteomic approach, we found that the regulators of inflammation and carboxypeptidase inhibitor, galectin-3 (LEG3; spot 58,59) and latexin (LXN; spot 54), were increased in the LC at day 14 post SCI (Table 2). By Western blot analysis, an increase in LEG3, but not LXN, was found along the longer survival time points (Figure 3). Cathepsin D (CATD), one of lysosomal enzymes enriched in macrophages, was also increased in the LC at day 14 post SCI (Table 2 and Figure 3). Furthermore, immuofluorescence indicated that Iba-1+ microglia were accumulated in the proximal site to the LC (Figure 5A). Moreover, CD11b (Mac-1) or CD49f-positive macrophages were observed in the LC (Figure 5B, C) and the proximal area to the LC at day 14 post SCI (data not shown).

Bottom Line: Our results showed a decline in catalase (CAT) and Mn-superoxide dismutase (MnSOD) found at day 14 after SCI.Delayed treatment with chondroitinase ABC (chABC) at day 3 post SCI improved the hindlimb locomotion in SCI rats.Our findings demonstrate that the differential expression in proteins related to signal transduction, oxidoreduction and stress contribute to extensive inflammation, causing time-dependent spread of tissue damage after severe SCI.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT

Background: Traumatic spinal cord injury (SCI) forms a disadvantageous microenvironment for tissue repair at the lesion site. To consider an appropriate time window for giving a promising therapeutic treatment for subacute and chronic SCI, global changes of proteins in the injured center at the longer survival time points after SCI remains to be elucidated.

Methods: Through two-dimensional electrophoresis (2DE)-based proteome analysis and western blotting, we examined the differential expression of the soluble proteins isolated from the lesion center (LC) at day 1 (acute) and day 14 (subacute) after a severe contusive injury to the thoracic spinal cord at segment 10. In situ apoptotic analysis was used to examine cell apoptosis in injured spinal cord after adenoviral gene transfer of antioxidant enzymes. In addition, administration of chondroitinase ABC (chABC) was performed to analyze hindlimb locomotor recovery in rats with SCI using Basso, Beattie and Bresnahan (BBB) locomotor rating scale.

Results: Our results showed a decline in catalase (CAT) and Mn-superoxide dismutase (MnSOD) found at day 14 after SCI. Accordingly, gene transfer of SOD was introduced in the injured spinal cord and found to attenuate cell apoptosis. Galectin-3, β-actin, actin regulatory protein (CAPG), and F-actin-capping protein subunit β (CAPZB) at day 14 were increased when compared to that detected at day 1 after SCI or in sham-operated control. Indeed, the accumulation of β-actin+ immune cells was observed in the LC at day 14 post SCI, while most of reactive astrocytes were surrounding the lesion center. In addition, chondroitin sulfate proteoglycans (CSPG)-related proteins with 40-kDa was detected in the LC at day 3-14 post SCI. Delayed treatment with chondroitinase ABC (chABC) at day 3 post SCI improved the hindlimb locomotion in SCI rats.

Conclusions: Our findings demonstrate that the differential expression in proteins related to signal transduction, oxidoreduction and stress contribute to extensive inflammation, causing time-dependent spread of tissue damage after severe SCI. The interventions by supplement of anti-oxidant enzymes right after SCI or delayed administration with chABC can facilitate spinal neural cell survival and tissue repair.

Show MeSH
Related in: MedlinePlus