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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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Photomicrographs of the morphological characterization of apoptotic neurons and astrocytes. A-D: photomicrographs of TUNEL and immunohistochemical double-stained sections 2 mm rostral from the epicenter in the ventral horn of injured spinal cords removed at 24 h post-SCI. A and B: TUNEL and NSE double-stained TUNEL-positive neurons at lower (A) and higher (B) magnifications. The arrowheads indicate TUNEL-positive nuclei in NSE-positive neurons. C and D: TUNEL and GFAP double-stained TUNEL-positive astrocyte at lower (C) and higher (D) magnifications. The arrows indicate TUNEL-positive nuclei in GFAP-positive astrocytes. E-J: photomicrographs of active caspase-3 and immuno-fluorescence double-stained sections 2.05 mm rostral from the epicenter in the ventral gray matter of injured spinal cords removed at 12 h post-SCI. E-G: p20 fragment and GFAP double-stained active caspase-3 with active astrocytes. E: active astrocytes (GFAP-positive, arrows); F: the same astrocytes stained for p20 (arrows). G: immuno-colocalization of E and F, showing caspase-3-positive astrocytes (arrows, yellow). H-J: p20 fragment and NSE double-stained neurons. H: NSE-positive neurons (arrowhead); I: the same neurons were also p20-positive (arrowhead). J: immuno-colocalization of H and I showing caspase-3-positive neurons (arrowhead, yellow). Scale bar = 100 μm.
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Figure 3: Photomicrographs of the morphological characterization of apoptotic neurons and astrocytes. A-D: photomicrographs of TUNEL and immunohistochemical double-stained sections 2 mm rostral from the epicenter in the ventral horn of injured spinal cords removed at 24 h post-SCI. A and B: TUNEL and NSE double-stained TUNEL-positive neurons at lower (A) and higher (B) magnifications. The arrowheads indicate TUNEL-positive nuclei in NSE-positive neurons. C and D: TUNEL and GFAP double-stained TUNEL-positive astrocyte at lower (C) and higher (D) magnifications. The arrows indicate TUNEL-positive nuclei in GFAP-positive astrocytes. E-J: photomicrographs of active caspase-3 and immuno-fluorescence double-stained sections 2.05 mm rostral from the epicenter in the ventral gray matter of injured spinal cords removed at 12 h post-SCI. E-G: p20 fragment and GFAP double-stained active caspase-3 with active astrocytes. E: active astrocytes (GFAP-positive, arrows); F: the same astrocytes stained for p20 (arrows). G: immuno-colocalization of E and F, showing caspase-3-positive astrocytes (arrows, yellow). H-J: p20 fragment and NSE double-stained neurons. H: NSE-positive neurons (arrowhead); I: the same neurons were also p20-positive (arrowhead). J: immuno-colocalization of H and I showing caspase-3-positive neurons (arrowhead, yellow). Scale bar = 100 μm.

Mentions: Figure 3 illustrates the photomicrographs of the morphological characterization of apoptosis in neurons and astrocytes. DNA fragmentation is an event of apoptosis and TUNEL stains the fragmented DNA in the nuclei. Anti-NSE and anti-GFAP immunohistochemical staining identifies neurons and reactive astrocytes, respectively. A to D in Figure 3 show the photomicrographs of TUNEL and NSE or GFAP immunohistochemically double-stained sections in the ventral horn of the gray matter in an injured spinal cord sections 2 mm rostral from the epicenter removed at 24 h post-SCI. The arrowheads in B indicate TUNEL-positive nuclei in NSE-positive neurons. The arrows in D indicate TUNEL-positive nuclei in GFAP-positive astrocytes. These double-stained spinal cord sections localize the apoptotic DNA fragmentation in the nuclei of neurons (A and B) and astrocytes (C and D). This is the morphological indication of neuronal and glial apoptosis. Since caspase-3 activation occurs prior to DNA fragmentation, the injured spinal cord was removed at 12 h post-SCI in this study for active caspase-3 staining. E to J in Figure 3 show the photomicrographs of active caspase-3 and NSE or GFAP immuno-fluorescence double-stained apoptotic astrocytes (E - G) and neurons (H - J), respectively, in the spinal cord sections 2.05 mm rostral from the epicenter removed at 12 h post-SCI. E to G show double immuno-fluorescence-stained sections with antibodies against an active fragment p20 of caspase-3 and anti-GFAP. E shows astrocytes (GFAP-positive, arrows) and F shows the same astrocytes stained for p20 (arrows). G is the colocalization of E and F, showing caspase-3-positive astrocytes (arrows). H to J show double immuno-fluorescence-stained neurons. H shows NSE-positive neurons (arrow head). I shows that the same neurons are also p20-positive (arrow head). J is colocalization of H and I, showing active caspase-3-positive neurons (arrow head). These double immuno-fluorescence staining co-localized caspase-3 activation in the neurons and astrocytes and provided another morphological indication of neuronal and glial apoptosis.


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)

Photomicrographs of the morphological characterization of apoptotic neurons and astrocytes. A-D: photomicrographs of TUNEL and immunohistochemical double-stained sections 2 mm rostral from the epicenter in the ventral horn of injured spinal cords removed at 24 h post-SCI. A and B: TUNEL and NSE double-stained TUNEL-positive neurons at lower (A) and higher (B) magnifications. The arrowheads indicate TUNEL-positive nuclei in NSE-positive neurons. C and D: TUNEL and GFAP double-stained TUNEL-positive astrocyte at lower (C) and higher (D) magnifications. The arrows indicate TUNEL-positive nuclei in GFAP-positive astrocytes. E-J: photomicrographs of active caspase-3 and immuno-fluorescence double-stained sections 2.05 mm rostral from the epicenter in the ventral gray matter of injured spinal cords removed at 12 h post-SCI. E-G: p20 fragment and GFAP double-stained active caspase-3 with active astrocytes. E: active astrocytes (GFAP-positive, arrows); F: the same astrocytes stained for p20 (arrows). G: immuno-colocalization of E and F, showing caspase-3-positive astrocytes (arrows, yellow). H-J: p20 fragment and NSE double-stained neurons. H: NSE-positive neurons (arrowhead); I: the same neurons were also p20-positive (arrowhead). J: immuno-colocalization of H and I showing caspase-3-positive neurons (arrowhead, yellow). Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924333&req=5

Figure 3: Photomicrographs of the morphological characterization of apoptotic neurons and astrocytes. A-D: photomicrographs of TUNEL and immunohistochemical double-stained sections 2 mm rostral from the epicenter in the ventral horn of injured spinal cords removed at 24 h post-SCI. A and B: TUNEL and NSE double-stained TUNEL-positive neurons at lower (A) and higher (B) magnifications. The arrowheads indicate TUNEL-positive nuclei in NSE-positive neurons. C and D: TUNEL and GFAP double-stained TUNEL-positive astrocyte at lower (C) and higher (D) magnifications. The arrows indicate TUNEL-positive nuclei in GFAP-positive astrocytes. E-J: photomicrographs of active caspase-3 and immuno-fluorescence double-stained sections 2.05 mm rostral from the epicenter in the ventral gray matter of injured spinal cords removed at 12 h post-SCI. E-G: p20 fragment and GFAP double-stained active caspase-3 with active astrocytes. E: active astrocytes (GFAP-positive, arrows); F: the same astrocytes stained for p20 (arrows). G: immuno-colocalization of E and F, showing caspase-3-positive astrocytes (arrows, yellow). H-J: p20 fragment and NSE double-stained neurons. H: NSE-positive neurons (arrowhead); I: the same neurons were also p20-positive (arrowhead). J: immuno-colocalization of H and I showing caspase-3-positive neurons (arrowhead, yellow). Scale bar = 100 μm.
Mentions: Figure 3 illustrates the photomicrographs of the morphological characterization of apoptosis in neurons and astrocytes. DNA fragmentation is an event of apoptosis and TUNEL stains the fragmented DNA in the nuclei. Anti-NSE and anti-GFAP immunohistochemical staining identifies neurons and reactive astrocytes, respectively. A to D in Figure 3 show the photomicrographs of TUNEL and NSE or GFAP immunohistochemically double-stained sections in the ventral horn of the gray matter in an injured spinal cord sections 2 mm rostral from the epicenter removed at 24 h post-SCI. The arrowheads in B indicate TUNEL-positive nuclei in NSE-positive neurons. The arrows in D indicate TUNEL-positive nuclei in GFAP-positive astrocytes. These double-stained spinal cord sections localize the apoptotic DNA fragmentation in the nuclei of neurons (A and B) and astrocytes (C and D). This is the morphological indication of neuronal and glial apoptosis. Since caspase-3 activation occurs prior to DNA fragmentation, the injured spinal cord was removed at 12 h post-SCI in this study for active caspase-3 staining. E to J in Figure 3 show the photomicrographs of active caspase-3 and NSE or GFAP immuno-fluorescence double-stained apoptotic astrocytes (E - G) and neurons (H - J), respectively, in the spinal cord sections 2.05 mm rostral from the epicenter removed at 12 h post-SCI. E to G show double immuno-fluorescence-stained sections with antibodies against an active fragment p20 of caspase-3 and anti-GFAP. E shows astrocytes (GFAP-positive, arrows) and F shows the same astrocytes stained for p20 (arrows). G is the colocalization of E and F, showing caspase-3-positive astrocytes (arrows). H to J show double immuno-fluorescence-stained neurons. H shows NSE-positive neurons (arrow head). I shows that the same neurons are also p20-positive (arrow head). J is colocalization of H and I, showing active caspase-3-positive neurons (arrow head). These double immuno-fluorescence staining co-localized caspase-3 activation in the neurons and astrocytes and provided another morphological indication of neuronal and glial apoptosis.

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