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The temporal and spatial profiles of cell loss following experimental spinal cord injury: effect of antioxidant therapy on cell death and functional recovery.

Ling X, Bao F, Qian H, Liu D - BMC Neurosci (2013)

Bottom Line: Intraperitoneal treatment with MnTBAP + nitro-L-arginine significantly reduced motoneuron and cell loss and apoptosis in the gray and white matter compared with the vehicle-treated group.MnTBAP alone significantly reduced the number of apoptotic cells and improved functional recovery as evaluated by three behavioral tests.Our demonstration that apoptosis follows SCI and that MnTBAP alone or MnTBAP + nitro-L-arginine significantly reduces apoptosis correlates SCI-induced apoptosis with RS overproduction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, University of Texas Medical Branch, 301 University Blvd,, Rt, 0881, Galveston, TX 77555-0881, USA. dliu@utmb.edu.

ABSTRACT

Background: Traumatic spinal cord injury (SCI)-induced overproduction of endogenous deleterious substances triggers secondary cell death to spread damage beyond the initial injury site. Substantial experimental evidence supports reactive species (RS) as important mediators of secondary cell death after SCI. This study established quantitative temporal and spatial profiles of cell loss, characterized apoptosis, and evaluated the effectiveness of a broad spectrum RS scavenger - Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) and a combination of MnTBAP plus nitro-L-arginine to prevent cell loss and neurological dysfunction following contusion SCI to the rat spinal cord.

Results: By counting the number of surviving cells in spinal cord sections removed at 1, 6, 12, 24, 48, 72 h and 1 week post-SCI and at 0 - 4 mm from the epicenter, the temporal and spatial profiles of motoneuron and glia loss were established. Motoneurons continued to disappear over a week and the losses decreased with increasing distance from the epicenter. Significant glia loss peaked at 24 to 48 h post-SCI, but only at sections 0-1.5 mm from the epicenter. Apoptosis of neurons, motoneurons and astrocytes was characterized morphologically by double immuno-staining with cell-specific markers and apoptosis indicators and confirmed by transmission electron microscopy. DNA laddering, ELISA quantitation and caspase-3 activation in the spinal cord tissue indicated more intense DNA fragments and greater caspase-3 activation in the epicenter than at 1 and 2 cm away from the epicenter or the sham-operated sections. Intraperitoneal treatment with MnTBAP + nitro-L-arginine significantly reduced motoneuron and cell loss and apoptosis in the gray and white matter compared with the vehicle-treated group. MnTBAP alone significantly reduced the number of apoptotic cells and improved functional recovery as evaluated by three behavioral tests.

Conclusions: Our temporal and spatial profiles of cell loss provide data bases for determining the time and location for pharmacological intervention. Our demonstration that apoptosis follows SCI and that MnTBAP alone or MnTBAP + nitro-L-arginine significantly reduces apoptosis correlates SCI-induced apoptosis with RS overproduction. MnTBAP significantly improved functional recovery, which strongly supports the important role of antioxidant therapy in treating SCI and the candidacy of MnTBAP for such treatment.

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Related in: MedlinePlus

Biochemical evidence of apoptosis. The spinal cord was removed at 48 h post-SCI by being frozen in situ with liquid nitrogen and divided into three sections of equal length (1 cm each) with section 1 centered at the epicenter and sections 2 and 3 centered at 1 and 2 cm caudal to the epicenter for DNA laddering and ELISA as described in the Methods. A: The laddering of DNA fragments in the spinal cord tissue. Lane 1, standard DNA marker; lane 2, the tissue obtained from the epicenter; lanes 3 and 4, tissue centered 1 and 2 cm away from the epicenter, respectively; and lane 5, tissue from a sham-operated rat. The fragmented DNA shown in each lane was obtained from the corresponding sections of one rat (60 mg wet weight tissue). The results are representative of four separate experiments. B: Quantitative analysis of fragmented DNA by ELISA with 3 sections at epicenter, 1 and 2 cm caudal from the epicenter. Each sample for ELISA analysis equaled 2 mg wet weight tissue. Error bars: mean ± SEM. Both A and B show significantly decreased DNA fragmentation with an increase in distance from the epicenter. C: Caspase-3 activation analyzed at 4 h post-SCI. The injured cord was divided into two sections with section 1 centered at the injury epicenter and section 2 centered 1 cm from the epicenter. Error bars: mean ± SEM. The caspase-3-like protease activity was significantly higher at the injury epicenter compared to 1 cm from the epicenter and sham control.
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Figure 6: Biochemical evidence of apoptosis. The spinal cord was removed at 48 h post-SCI by being frozen in situ with liquid nitrogen and divided into three sections of equal length (1 cm each) with section 1 centered at the epicenter and sections 2 and 3 centered at 1 and 2 cm caudal to the epicenter for DNA laddering and ELISA as described in the Methods. A: The laddering of DNA fragments in the spinal cord tissue. Lane 1, standard DNA marker; lane 2, the tissue obtained from the epicenter; lanes 3 and 4, tissue centered 1 and 2 cm away from the epicenter, respectively; and lane 5, tissue from a sham-operated rat. The fragmented DNA shown in each lane was obtained from the corresponding sections of one rat (60 mg wet weight tissue). The results are representative of four separate experiments. B: Quantitative analysis of fragmented DNA by ELISA with 3 sections at epicenter, 1 and 2 cm caudal from the epicenter. Each sample for ELISA analysis equaled 2 mg wet weight tissue. Error bars: mean ± SEM. Both A and B show significantly decreased DNA fragmentation with an increase in distance from the epicenter. C: Caspase-3 activation analyzed at 4 h post-SCI. The injured cord was divided into two sections with section 1 centered at the injury epicenter and section 2 centered 1 cm from the epicenter. Error bars: mean ± SEM. The caspase-3-like protease activity was significantly higher at the injury epicenter compared to 1 cm from the epicenter and sham control.

Mentions: Figure 6 represents the results of our biochemical analysis. Figure 6A shows autoradiographs of DNA fragments analyzed by agarose gel electrophoresis from the cords extracted at 48 h post-SCI (lanes 2 – 4) and the sham-operated control (lane 5). Fragmented DNA displayed a ladder-like electrophoretic pattern. The intensity of the ladder was higher in lane 2 (epicenter) than in lane 3 (section 2 – 1 cm from the epicenter). No fragmented DNA bands were observed in lane 4 (section 3 – 2 cm from the epicenter). The sham-operated sample (lane 5) did not show any detectable fragmentation. DNA fragmentation was quantitated by ELISA (Figure 6B). The cellular DNA isolated from three sections of injured spinal cord tissue showed a significantly higher absorbance at 405 nm than did the three corresponding sections in the sham-operated rats (n = 6, p < 0.001, RMANOVA followed by all pairwise multiple comparison with the Bonferroni correction). The average increases in the three injured sections were 2- to 10-fold that of corresponding sham controls. Fragmented DNA in the injured tissue was more abundant in the epicenter (section 1) than in 1 (section 2) and 2 (section 3) cm from the epicenter (p < 0.001), and in 1 cm than in 2 cm caudal from the epicenter (p = 0.01). The decline over distance from the injury site measured by ELISA (Figure 6B) was comparable to the decline in intensity of DNA laddering over the same distance (Figure 6A). Figure 6C shows caspase-3 activation at 4 h post-SCI. The caspase-3-like protease activity was measured as described in the Methods. The caspase-3-like protease activity was significantly higher in the epicenter (section 1) of the cords than at 1 cm from the epicenter (section 2) in the injured cords (n = 5) or in the sham control (n = 4, p = 0.03 for both). Results for the sham control and 1 cm from the epicenter did not differ significantly.


The temporal and spatial profiles of cell loss following experimental spinal cord injury: effect of antioxidant therapy on cell death and functional recovery.

Ling X, Bao F, Qian H, Liu D - BMC Neurosci (2013)

Biochemical evidence of apoptosis. The spinal cord was removed at 48 h post-SCI by being frozen in situ with liquid nitrogen and divided into three sections of equal length (1 cm each) with section 1 centered at the epicenter and sections 2 and 3 centered at 1 and 2 cm caudal to the epicenter for DNA laddering and ELISA as described in the Methods. A: The laddering of DNA fragments in the spinal cord tissue. Lane 1, standard DNA marker; lane 2, the tissue obtained from the epicenter; lanes 3 and 4, tissue centered 1 and 2 cm away from the epicenter, respectively; and lane 5, tissue from a sham-operated rat. The fragmented DNA shown in each lane was obtained from the corresponding sections of one rat (60 mg wet weight tissue). The results are representative of four separate experiments. B: Quantitative analysis of fragmented DNA by ELISA with 3 sections at epicenter, 1 and 2 cm caudal from the epicenter. Each sample for ELISA analysis equaled 2 mg wet weight tissue. Error bars: mean ± SEM. Both A and B show significantly decreased DNA fragmentation with an increase in distance from the epicenter. C: Caspase-3 activation analyzed at 4 h post-SCI. The injured cord was divided into two sections with section 1 centered at the injury epicenter and section 2 centered 1 cm from the epicenter. Error bars: mean ± SEM. The caspase-3-like protease activity was significantly higher at the injury epicenter compared to 1 cm from the epicenter and sham control.
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Related In: Results  -  Collection

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Figure 6: Biochemical evidence of apoptosis. The spinal cord was removed at 48 h post-SCI by being frozen in situ with liquid nitrogen and divided into three sections of equal length (1 cm each) with section 1 centered at the epicenter and sections 2 and 3 centered at 1 and 2 cm caudal to the epicenter for DNA laddering and ELISA as described in the Methods. A: The laddering of DNA fragments in the spinal cord tissue. Lane 1, standard DNA marker; lane 2, the tissue obtained from the epicenter; lanes 3 and 4, tissue centered 1 and 2 cm away from the epicenter, respectively; and lane 5, tissue from a sham-operated rat. The fragmented DNA shown in each lane was obtained from the corresponding sections of one rat (60 mg wet weight tissue). The results are representative of four separate experiments. B: Quantitative analysis of fragmented DNA by ELISA with 3 sections at epicenter, 1 and 2 cm caudal from the epicenter. Each sample for ELISA analysis equaled 2 mg wet weight tissue. Error bars: mean ± SEM. Both A and B show significantly decreased DNA fragmentation with an increase in distance from the epicenter. C: Caspase-3 activation analyzed at 4 h post-SCI. The injured cord was divided into two sections with section 1 centered at the injury epicenter and section 2 centered 1 cm from the epicenter. Error bars: mean ± SEM. The caspase-3-like protease activity was significantly higher at the injury epicenter compared to 1 cm from the epicenter and sham control.
Mentions: Figure 6 represents the results of our biochemical analysis. Figure 6A shows autoradiographs of DNA fragments analyzed by agarose gel electrophoresis from the cords extracted at 48 h post-SCI (lanes 2 – 4) and the sham-operated control (lane 5). Fragmented DNA displayed a ladder-like electrophoretic pattern. The intensity of the ladder was higher in lane 2 (epicenter) than in lane 3 (section 2 – 1 cm from the epicenter). No fragmented DNA bands were observed in lane 4 (section 3 – 2 cm from the epicenter). The sham-operated sample (lane 5) did not show any detectable fragmentation. DNA fragmentation was quantitated by ELISA (Figure 6B). The cellular DNA isolated from three sections of injured spinal cord tissue showed a significantly higher absorbance at 405 nm than did the three corresponding sections in the sham-operated rats (n = 6, p < 0.001, RMANOVA followed by all pairwise multiple comparison with the Bonferroni correction). The average increases in the three injured sections were 2- to 10-fold that of corresponding sham controls. Fragmented DNA in the injured tissue was more abundant in the epicenter (section 1) than in 1 (section 2) and 2 (section 3) cm from the epicenter (p < 0.001), and in 1 cm than in 2 cm caudal from the epicenter (p = 0.01). The decline over distance from the injury site measured by ELISA (Figure 6B) was comparable to the decline in intensity of DNA laddering over the same distance (Figure 6A). Figure 6C shows caspase-3 activation at 4 h post-SCI. The caspase-3-like protease activity was measured as described in the Methods. The caspase-3-like protease activity was significantly higher in the epicenter (section 1) of the cords than at 1 cm from the epicenter (section 2) in the injured cords (n = 5) or in the sham control (n = 4, p = 0.03 for both). Results for the sham control and 1 cm from the epicenter did not differ significantly.

Bottom Line: Intraperitoneal treatment with MnTBAP + nitro-L-arginine significantly reduced motoneuron and cell loss and apoptosis in the gray and white matter compared with the vehicle-treated group.MnTBAP alone significantly reduced the number of apoptotic cells and improved functional recovery as evaluated by three behavioral tests.Our demonstration that apoptosis follows SCI and that MnTBAP alone or MnTBAP + nitro-L-arginine significantly reduces apoptosis correlates SCI-induced apoptosis with RS overproduction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurology, University of Texas Medical Branch, 301 University Blvd,, Rt, 0881, Galveston, TX 77555-0881, USA. dliu@utmb.edu.

ABSTRACT

Background: Traumatic spinal cord injury (SCI)-induced overproduction of endogenous deleterious substances triggers secondary cell death to spread damage beyond the initial injury site. Substantial experimental evidence supports reactive species (RS) as important mediators of secondary cell death after SCI. This study established quantitative temporal and spatial profiles of cell loss, characterized apoptosis, and evaluated the effectiveness of a broad spectrum RS scavenger - Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) and a combination of MnTBAP plus nitro-L-arginine to prevent cell loss and neurological dysfunction following contusion SCI to the rat spinal cord.

Results: By counting the number of surviving cells in spinal cord sections removed at 1, 6, 12, 24, 48, 72 h and 1 week post-SCI and at 0 - 4 mm from the epicenter, the temporal and spatial profiles of motoneuron and glia loss were established. Motoneurons continued to disappear over a week and the losses decreased with increasing distance from the epicenter. Significant glia loss peaked at 24 to 48 h post-SCI, but only at sections 0-1.5 mm from the epicenter. Apoptosis of neurons, motoneurons and astrocytes was characterized morphologically by double immuno-staining with cell-specific markers and apoptosis indicators and confirmed by transmission electron microscopy. DNA laddering, ELISA quantitation and caspase-3 activation in the spinal cord tissue indicated more intense DNA fragments and greater caspase-3 activation in the epicenter than at 1 and 2 cm away from the epicenter or the sham-operated sections. Intraperitoneal treatment with MnTBAP + nitro-L-arginine significantly reduced motoneuron and cell loss and apoptosis in the gray and white matter compared with the vehicle-treated group. MnTBAP alone significantly reduced the number of apoptotic cells and improved functional recovery as evaluated by three behavioral tests.

Conclusions: Our temporal and spatial profiles of cell loss provide data bases for determining the time and location for pharmacological intervention. Our demonstration that apoptosis follows SCI and that MnTBAP alone or MnTBAP + nitro-L-arginine significantly reduces apoptosis correlates SCI-induced apoptosis with RS overproduction. MnTBAP significantly improved functional recovery, which strongly supports the important role of antioxidant therapy in treating SCI and the candidacy of MnTBAP for such treatment.

Show MeSH
Related in: MedlinePlus