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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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MnTBAP + L-NA reduced SCI-induced apoptosis. Spinal cord sections were obtained at 12 h post SCI as in Figure 7. Upper: photomicrographs of TUNEL-stained sections at 2 mm rostral from the epicenter in the gray matter of the cord. A-C: lower magnification and D-F: higher magnification of A-C. A and D: sham control; B and E: injured section; C and F: injured section treated with MnTBAP + L-NA. Treatment by MnTBAP + L-NA clearly reduced the number of TUNEL-positive cells. The TUNEL-positive cells at the sections 2 mm from the epicenter were counted in both the gray and the white matter and the counts compared between the combination-treated and vehicle-treated groups (lower panel). a: Number of TUNEL-positive cells in the gray matter. b: Number of TUNEL-positive cells in the white matter. Error bars, mean ± SEM. Asterisk indicates a significant difference between the two treatment groups (p < 0.05). The combination of MnTBAP + L-NA significantly reduced the number of TUNEL-positive cells in both the gray and the white matter.
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Figure 8: MnTBAP + L-NA reduced SCI-induced apoptosis. Spinal cord sections were obtained at 12 h post SCI as in Figure 7. Upper: photomicrographs of TUNEL-stained sections at 2 mm rostral from the epicenter in the gray matter of the cord. A-C: lower magnification and D-F: higher magnification of A-C. A and D: sham control; B and E: injured section; C and F: injured section treated with MnTBAP + L-NA. Treatment by MnTBAP + L-NA clearly reduced the number of TUNEL-positive cells. The TUNEL-positive cells at the sections 2 mm from the epicenter were counted in both the gray and the white matter and the counts compared between the combination-treated and vehicle-treated groups (lower panel). a: Number of TUNEL-positive cells in the gray matter. b: Number of TUNEL-positive cells in the white matter. Error bars, mean ± SEM. Asterisk indicates a significant difference between the two treatment groups (p < 0.05). The combination of MnTBAP + L-NA significantly reduced the number of TUNEL-positive cells in both the gray and the white matter.

Mentions: Since the number of TUNEL-positive cells in the gray matter peaked from 12 to 48 h post-SCI [43], the effect of MnTBAP + L-NA on apoptosis was evaluated at 12 h post-SCI by counting the TUNEL-positive cells in the gray and white matter in the vehicle- and combination-treated sections as described above (Figure 8). The upper panel of Figure 8 provides photomicrographs of TUNEL-stained sections 2 mm rostral from the epicenter in the gray matter of the cord at 12 h post-SCI. The sham control sections showed no TUNEL-positive cells in the gray matter of the cord (A, D). In the SCI group (B, E), many TUNEL-positive cells appeared. Treatment with the combination clearly reduced the number of TUNEL-positive cells (C, F). The cells in the sections 2 mm rostral from the epicenter in the vehicle-treated (n = 5) and the combination-treated groups (n = 6) were counted and the counts compared using unpaired t-test. The quantitative comparison demonstrated that the MnTBAP + L-NA treatment significantly (p = 0.01) reduced the number of TUNEL-positive cells in the gray (Figure 8a) and white (Figure 8b) matter, suggesting that RS contribute to apoptotic DNA fragmentation following SCI.


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)

MnTBAP + L-NA reduced SCI-induced apoptosis. Spinal cord sections were obtained at 12 h post SCI as in Figure 7. Upper: photomicrographs of TUNEL-stained sections at 2 mm rostral from the epicenter in the gray matter of the cord. A-C: lower magnification and D-F: higher magnification of A-C. A and D: sham control; B and E: injured section; C and F: injured section treated with MnTBAP + L-NA. Treatment by MnTBAP + L-NA clearly reduced the number of TUNEL-positive cells. The TUNEL-positive cells at the sections 2 mm from the epicenter were counted in both the gray and the white matter and the counts compared between the combination-treated and vehicle-treated groups (lower panel). a: Number of TUNEL-positive cells in the gray matter. b: Number of TUNEL-positive cells in the white matter. Error bars, mean ± SEM. Asterisk indicates a significant difference between the two treatment groups (p < 0.05). The combination of MnTBAP + L-NA significantly reduced the number of TUNEL-positive cells in both the gray and the white matter.
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Related In: Results  -  Collection

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Figure 8: MnTBAP + L-NA reduced SCI-induced apoptosis. Spinal cord sections were obtained at 12 h post SCI as in Figure 7. Upper: photomicrographs of TUNEL-stained sections at 2 mm rostral from the epicenter in the gray matter of the cord. A-C: lower magnification and D-F: higher magnification of A-C. A and D: sham control; B and E: injured section; C and F: injured section treated with MnTBAP + L-NA. Treatment by MnTBAP + L-NA clearly reduced the number of TUNEL-positive cells. The TUNEL-positive cells at the sections 2 mm from the epicenter were counted in both the gray and the white matter and the counts compared between the combination-treated and vehicle-treated groups (lower panel). a: Number of TUNEL-positive cells in the gray matter. b: Number of TUNEL-positive cells in the white matter. Error bars, mean ± SEM. Asterisk indicates a significant difference between the two treatment groups (p < 0.05). The combination of MnTBAP + L-NA significantly reduced the number of TUNEL-positive cells in both the gray and the white matter.
Mentions: Since the number of TUNEL-positive cells in the gray matter peaked from 12 to 48 h post-SCI [43], the effect of MnTBAP + L-NA on apoptosis was evaluated at 12 h post-SCI by counting the TUNEL-positive cells in the gray and white matter in the vehicle- and combination-treated sections as described above (Figure 8). The upper panel of Figure 8 provides photomicrographs of TUNEL-stained sections 2 mm rostral from the epicenter in the gray matter of the cord at 12 h post-SCI. The sham control sections showed no TUNEL-positive cells in the gray matter of the cord (A, D). In the SCI group (B, E), many TUNEL-positive cells appeared. Treatment with the combination clearly reduced the number of TUNEL-positive cells (C, F). The cells in the sections 2 mm rostral from the epicenter in the vehicle-treated (n = 5) and the combination-treated groups (n = 6) were counted and the counts compared using unpaired t-test. The quantitative comparison demonstrated that the MnTBAP + L-NA treatment significantly (p = 0.01) reduced the number of TUNEL-positive cells in the gray (Figure 8a) and white (Figure 8b) matter, suggesting that RS contribute to apoptotic DNA fragmentation following SCI.

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