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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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Time expression prolife of GFAP and CSPGs in the injured spinal cord. (A) The injured spinal cord tissue sections were collected at day 1, 7, and 14 after SCI. The tissue sections were subjected to immunofluorescence for GFAP (red). GFAP+ stellated cells (open arrows) were observed in the areas proximal to the lesion center (LC) or migrated into the LC at day 7 and 14 after SCI, and the GFAP+ cells were also immunoreactive with β-actin (insets). Note that cell debris with GFAP immunoreactivity (arrows) was detected in the LC at 1 day post SCI. Scale bar, 40 μm. (B). Total proteins were prepared from injured spinal cord tissues 0.5 or 1 mm rostral (r0.5 and r1) and caudal (c0.5 and c1) to the injury epicenter (LC) at day 31 post SCI (left panel). Sham-operated control was from animal only receiving laminectomy. Alternatively, the proteins were extracted from the lesion centers of spinal cord tissues at day 1, 3, 7 and 14 post SCI (right panel). The sample preparation was described in Materials and Methods. To examine the levels of CSPGs in the injured spinal cord tissues, western blotting was then performed using anti-chondroitin-4-sulfate antibody. Arrows indicate CSPGs or CSPG fragments with 40 kDa approximately.
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Figure 6: Time expression prolife of GFAP and CSPGs in the injured spinal cord. (A) The injured spinal cord tissue sections were collected at day 1, 7, and 14 after SCI. The tissue sections were subjected to immunofluorescence for GFAP (red). GFAP+ stellated cells (open arrows) were observed in the areas proximal to the lesion center (LC) or migrated into the LC at day 7 and 14 after SCI, and the GFAP+ cells were also immunoreactive with β-actin (insets). Note that cell debris with GFAP immunoreactivity (arrows) was detected in the LC at 1 day post SCI. Scale bar, 40 μm. (B). Total proteins were prepared from injured spinal cord tissues 0.5 or 1 mm rostral (r0.5 and r1) and caudal (c0.5 and c1) to the injury epicenter (LC) at day 31 post SCI (left panel). Sham-operated control was from animal only receiving laminectomy. Alternatively, the proteins were extracted from the lesion centers of spinal cord tissues at day 1, 3, 7 and 14 post SCI (right panel). The sample preparation was described in Materials and Methods. To examine the levels of CSPGs in the injured spinal cord tissues, western blotting was then performed using anti-chondroitin-4-sulfate antibody. Arrows indicate CSPGs or CSPG fragments with 40 kDa approximately.

Mentions: We noticed no change in the levels of astrocytic proteins, GS (spot 36 and 37) and GFAP (spot 87) in the LC at day 1 and 14 post SCI (Table 4). Immunofluorescence indicated that GFAP+ cell fragments were observed at the lesion site at day 1 post SCI (Figure 6A), while GFAP+ hypertrophic astrocytes were detected in the injury penumbra at day 7 and 14 post SCI. These GFAP+ cells were also colocalized to β-actin+ cells (Figure 6A, insets). In addition, we observed that few GFAP+ astrocytic processes invaded to the LC (Figure 6A) at day 14 post SCI. The results from immunofluorescence explain that comparable GFAP detected by proteome analysis in the LC at day 14 was derived from invading astrocytes, which is the pathophysiological event proposed in SCI [1]. Given the fact that glial scar is mainly formed by chondroitin sulfate proteoglycans (CSPGs) primarily produced by reactive astrocytes, the production of CSPGs at the different spinal cord tissue blocks was examined at day 31 after SCI. As shown in Figure 6B, there were differential levels of CSPGs detected in the spinal cord tissues rostral and caudal to the lesion center. However, CSPGs approximately corresponding to 40- kDa were only present in the LC. In addition, the 40-kDa CSPGs were initially detected in the LC at day 3, continued to be seen at day 7 and 14 post SCI (Figure 6B). Based on the spatial and temporal levels of 40-kDa CSPGs in the injured spinal cord, injection into the injured spinal cord with chABC at the different time points post SCI was performed. The hindlimb locomotor function was assessed every 2-3 days up to 31 days using BBB locomotor rating scale. Through the evaluation of behavior analysis, we found that administration of chABC right after SCI or at day 3 post SCI enhanced the hindlimb locomotion in rats with SCI (Figure 7A). However, at day 31 after SCI, BBB scores in rats receiving delayed treatment with chABC were higher than that observed in animals with acute treatment with chABC. Immunofluorescence showed that there were numerous neuronal fiber bundles with GAP-43-positive staining in the injured spinal cord receiving chABC immediately after SCI or by delayed treatment with chABC (Figure 7B, arrows), whereas only numerous fine fragmented neuronal fibers remained in the injured spinal cord without treatment (Figure 7B, arrowheads). In addition, when compared to GAP-43 immunostaining on the LC with acute chABC treatment, there was more elongated GAP-43-positivie neuronal fiber bundles present in the injured center of the spinal cord with delayed treatment by chABC (Figure 7B).


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)

Time expression prolife of GFAP and CSPGs in the injured spinal cord. (A) The injured spinal cord tissue sections were collected at day 1, 7, and 14 after SCI. The tissue sections were subjected to immunofluorescence for GFAP (red). GFAP+ stellated cells (open arrows) were observed in the areas proximal to the lesion center (LC) or migrated into the LC at day 7 and 14 after SCI, and the GFAP+ cells were also immunoreactive with β-actin (insets). Note that cell debris with GFAP immunoreactivity (arrows) was detected in the LC at 1 day post SCI. Scale bar, 40 μm. (B). Total proteins were prepared from injured spinal cord tissues 0.5 or 1 mm rostral (r0.5 and r1) and caudal (c0.5 and c1) to the injury epicenter (LC) at day 31 post SCI (left panel). Sham-operated control was from animal only receiving laminectomy. Alternatively, the proteins were extracted from the lesion centers of spinal cord tissues at day 1, 3, 7 and 14 post SCI (right panel). The sample preparation was described in Materials and Methods. To examine the levels of CSPGs in the injured spinal cord tissues, western blotting was then performed using anti-chondroitin-4-sulfate antibody. Arrows indicate CSPGs or CSPG fragments with 40 kDa approximately.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Time expression prolife of GFAP and CSPGs in the injured spinal cord. (A) The injured spinal cord tissue sections were collected at day 1, 7, and 14 after SCI. The tissue sections were subjected to immunofluorescence for GFAP (red). GFAP+ stellated cells (open arrows) were observed in the areas proximal to the lesion center (LC) or migrated into the LC at day 7 and 14 after SCI, and the GFAP+ cells were also immunoreactive with β-actin (insets). Note that cell debris with GFAP immunoreactivity (arrows) was detected in the LC at 1 day post SCI. Scale bar, 40 μm. (B). Total proteins were prepared from injured spinal cord tissues 0.5 or 1 mm rostral (r0.5 and r1) and caudal (c0.5 and c1) to the injury epicenter (LC) at day 31 post SCI (left panel). Sham-operated control was from animal only receiving laminectomy. Alternatively, the proteins were extracted from the lesion centers of spinal cord tissues at day 1, 3, 7 and 14 post SCI (right panel). The sample preparation was described in Materials and Methods. To examine the levels of CSPGs in the injured spinal cord tissues, western blotting was then performed using anti-chondroitin-4-sulfate antibody. Arrows indicate CSPGs or CSPG fragments with 40 kDa approximately.
Mentions: We noticed no change in the levels of astrocytic proteins, GS (spot 36 and 37) and GFAP (spot 87) in the LC at day 1 and 14 post SCI (Table 4). Immunofluorescence indicated that GFAP+ cell fragments were observed at the lesion site at day 1 post SCI (Figure 6A), while GFAP+ hypertrophic astrocytes were detected in the injury penumbra at day 7 and 14 post SCI. These GFAP+ cells were also colocalized to β-actin+ cells (Figure 6A, insets). In addition, we observed that few GFAP+ astrocytic processes invaded to the LC (Figure 6A) at day 14 post SCI. The results from immunofluorescence explain that comparable GFAP detected by proteome analysis in the LC at day 14 was derived from invading astrocytes, which is the pathophysiological event proposed in SCI [1]. Given the fact that glial scar is mainly formed by chondroitin sulfate proteoglycans (CSPGs) primarily produced by reactive astrocytes, the production of CSPGs at the different spinal cord tissue blocks was examined at day 31 after SCI. As shown in Figure 6B, there were differential levels of CSPGs detected in the spinal cord tissues rostral and caudal to the lesion center. However, CSPGs approximately corresponding to 40- kDa were only present in the LC. In addition, the 40-kDa CSPGs were initially detected in the LC at day 3, continued to be seen at day 7 and 14 post SCI (Figure 6B). Based on the spatial and temporal levels of 40-kDa CSPGs in the injured spinal cord, injection into the injured spinal cord with chABC at the different time points post SCI was performed. The hindlimb locomotor function was assessed every 2-3 days up to 31 days using BBB locomotor rating scale. Through the evaluation of behavior analysis, we found that administration of chABC right after SCI or at day 3 post SCI enhanced the hindlimb locomotion in rats with SCI (Figure 7A). However, at day 31 after SCI, BBB scores in rats receiving delayed treatment with chABC were higher than that observed in animals with acute treatment with chABC. Immunofluorescence showed that there were numerous neuronal fiber bundles with GAP-43-positive staining in the injured spinal cord receiving chABC immediately after SCI or by delayed treatment with chABC (Figure 7B, arrows), whereas only numerous fine fragmented neuronal fibers remained in the injured spinal cord without treatment (Figure 7B, arrowheads). In addition, when compared to GAP-43 immunostaining on the LC with acute chABC treatment, there was more elongated GAP-43-positivie neuronal fiber bundles present in the injured center of the spinal cord with delayed treatment by chABC (Figure 7B).

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