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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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Proteome analysis of the lesion center of the injured spinal cord. (A). The injured spinal cords were collected at day 1 (acute) and day 14 (subacute) after SCI. The lesion center (LC) with the length of 4-5 mm was dissected from the injured spinal cord tissues, and subjected to protein extraction for 2-DE. (B). Representative silver stained 2-DE gels show protein spots in the LC of the spinal cord derived from acute and subacute-SCI rats. Protein samples (200 μg) were loaded onto IPG strips (pH 3-10 Non-Linear) and then separated by a 10% SDS-PAGE gel. The gel was stained with silver stain and analyzed. Similar patterns of protein spots on the 2-DE were observed in six independent gels from three different sets of experiments. The spots on the gels were excised, trypsinized, and analyzed by MALDI-TOF-MS as described in Materials and Methods. Protein identification was obtained for 128 protein spots. There were 7 proteins which were biostatistically reduced in the LC at day 14. 12 proteins were found to be significantly upregulated in the LC at day 14 when compared to that detected at day 1 post SCI. Their protein identification and fold change in their expression levels were shown in Table 1 and 2.
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Figure 1: Proteome analysis of the lesion center of the injured spinal cord. (A). The injured spinal cords were collected at day 1 (acute) and day 14 (subacute) after SCI. The lesion center (LC) with the length of 4-5 mm was dissected from the injured spinal cord tissues, and subjected to protein extraction for 2-DE. (B). Representative silver stained 2-DE gels show protein spots in the LC of the spinal cord derived from acute and subacute-SCI rats. Protein samples (200 μg) were loaded onto IPG strips (pH 3-10 Non-Linear) and then separated by a 10% SDS-PAGE gel. The gel was stained with silver stain and analyzed. Similar patterns of protein spots on the 2-DE were observed in six independent gels from three different sets of experiments. The spots on the gels were excised, trypsinized, and analyzed by MALDI-TOF-MS as described in Materials and Methods. Protein identification was obtained for 128 protein spots. There were 7 proteins which were biostatistically reduced in the LC at day 14. 12 proteins were found to be significantly upregulated in the LC at day 14 when compared to that detected at day 1 post SCI. Their protein identification and fold change in their expression levels were shown in Table 1 and 2.

Mentions: The spinal cord tissues were dissected from the LC at day 1 (acute) or day 14 (subacute) post SCI (Figure 1A). Through 2-DE and subjected to MALDI-TOF analysis, we found that protein spots mainly appeared in the section of the pI values 3-10 and the molecular weight was approximately from 20-130 kDa (Figure 1B). An average of 222 protein spots were detected by Image Master 2D analysis software in the acute group and 238 protein spots in the subacute group (Figure 1B). Total 128 proteins were successfully identified through MALDI-TOF mass spectrometry and subsequent database searching (Tables 1, 2, 3 and 4). In comparison to the protein expression in the acute group, quantitative data indicated that the expression intensity of 7 or 12 proteins was biostatistically decreased (Table 1) or increased (Table 2) at least by 1.5-fold in the subacute phase, respectively. However, 42 proteins were considered to have less difference in their expression between day 1 and 14 post SCI (Table 3).


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)

Proteome analysis of the lesion center of the injured spinal cord. (A). The injured spinal cords were collected at day 1 (acute) and day 14 (subacute) after SCI. The lesion center (LC) with the length of 4-5 mm was dissected from the injured spinal cord tissues, and subjected to protein extraction for 2-DE. (B). Representative silver stained 2-DE gels show protein spots in the LC of the spinal cord derived from acute and subacute-SCI rats. Protein samples (200 μg) were loaded onto IPG strips (pH 3-10 Non-Linear) and then separated by a 10% SDS-PAGE gel. The gel was stained with silver stain and analyzed. Similar patterns of protein spots on the 2-DE were observed in six independent gels from three different sets of experiments. The spots on the gels were excised, trypsinized, and analyzed by MALDI-TOF-MS as described in Materials and Methods. Protein identification was obtained for 128 protein spots. There were 7 proteins which were biostatistically reduced in the LC at day 14. 12 proteins were found to be significantly upregulated in the LC at day 14 when compared to that detected at day 1 post SCI. Their protein identification and fold change in their expression levels were shown in Table 1 and 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040708&req=5

Figure 1: Proteome analysis of the lesion center of the injured spinal cord. (A). The injured spinal cords were collected at day 1 (acute) and day 14 (subacute) after SCI. The lesion center (LC) with the length of 4-5 mm was dissected from the injured spinal cord tissues, and subjected to protein extraction for 2-DE. (B). Representative silver stained 2-DE gels show protein spots in the LC of the spinal cord derived from acute and subacute-SCI rats. Protein samples (200 μg) were loaded onto IPG strips (pH 3-10 Non-Linear) and then separated by a 10% SDS-PAGE gel. The gel was stained with silver stain and analyzed. Similar patterns of protein spots on the 2-DE were observed in six independent gels from three different sets of experiments. The spots on the gels were excised, trypsinized, and analyzed by MALDI-TOF-MS as described in Materials and Methods. Protein identification was obtained for 128 protein spots. There were 7 proteins which were biostatistically reduced in the LC at day 14. 12 proteins were found to be significantly upregulated in the LC at day 14 when compared to that detected at day 1 post SCI. Their protein identification and fold change in their expression levels were shown in Table 1 and 2.
Mentions: The spinal cord tissues were dissected from the LC at day 1 (acute) or day 14 (subacute) post SCI (Figure 1A). Through 2-DE and subjected to MALDI-TOF analysis, we found that protein spots mainly appeared in the section of the pI values 3-10 and the molecular weight was approximately from 20-130 kDa (Figure 1B). An average of 222 protein spots were detected by Image Master 2D analysis software in the acute group and 238 protein spots in the subacute group (Figure 1B). Total 128 proteins were successfully identified through MALDI-TOF mass spectrometry and subsequent database searching (Tables 1, 2, 3 and 4). In comparison to the protein expression in the acute group, quantitative data indicated that the expression intensity of 7 or 12 proteins was biostatistically decreased (Table 1) or increased (Table 2) at least by 1.5-fold in the subacute phase, respectively. However, 42 proteins were considered to have less difference in their expression between day 1 and 14 post SCI (Table 3).

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