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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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TEM confirmation of neuronal and glial apoptosis. The tissue for TEM was obtained at 24 h post-SCI from layer VIII of the ventral horn of the cord and the 2–2.5 mm rostral from the epicenter were processed for TEM as described in the Methods. A: a normal neuron from sham control (×14,300). B: a degenerating neuronal axon (×3,300). C (×24,475); D (×14,300); E (×42,625); F (×18,150): different stages of apoptotic neurons and glial cells. Symbols: N-Nucleus, M-Mitochondria, R- Rough endoplasmic reticulum, G-Golgi complex, Ch-Chromatin.
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Figure 5: TEM confirmation of neuronal and glial apoptosis. The tissue for TEM was obtained at 24 h post-SCI from layer VIII of the ventral horn of the cord and the 2–2.5 mm rostral from the epicenter were processed for TEM as described in the Methods. A: a normal neuron from sham control (×14,300). B: a degenerating neuronal axon (×3,300). C (×24,475); D (×14,300); E (×42,625); F (×18,150): different stages of apoptotic neurons and glial cells. Symbols: N-Nucleus, M-Mitochondria, R- Rough endoplasmic reticulum, G-Golgi complex, Ch-Chromatin.

Mentions: SCI-induced apoptosis was confirmed by TEM identification based on the specific morphology of apoptosis. Figure 5 illustrates the ultra-fine structural changes in apoptotic cells in layer VIII of the ventral horn in the gray matter of the spinal cord sections at 2-2.5 mm rostral from the epicenter removed at 24 h post-SCI (50 g.cm). Figure 5A, a sham control section 2 mm rostral from the epicenter, shows a normal neuron with a large, euchromatic (pale-staining) nucleus (N), abundant rough endoplasmic reticulum (R), mitochondria (M), and Golgi complexes (G) in the cytoplasm. Figure 5B-F show different stages of apoptosis and degeneration from injured animals. B shows a degenerating swollen axon (arrow) with vacuoles. C-F shows very early to late stages of apoptotic degeneration of neuronal and glial cells. C shows a very early stage apoptotic neuron with condensed chromatin (Ch) in the rim of the nucleus (arrow) and unchanged organelle in the cytoplasm. D shows a normal glial cell (arrow) with a smaller cell body and heterochromatic nucleus stained more intensely than the nucleus of the neuron. An apoptotic glial cell near the normal glial cell (arrow head) is shrinking, becoming denser and the chromatin undergoing condensation to form an irregular dense mass while the organelles are becoming indiscernible. E shows the early stage of an apoptotic neuron with condensed chromatin accumulated mainly at the nuclear rim; the organelles are intact. F represents a nucleus which is fragmented and has formed highly condensed apoptotic bodies (arrow head). A phagocyte (arrow) is shown engulfing the apoptotic body.


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)

TEM confirmation of neuronal and glial apoptosis. The tissue for TEM was obtained at 24 h post-SCI from layer VIII of the ventral horn of the cord and the 2–2.5 mm rostral from the epicenter were processed for TEM as described in the Methods. A: a normal neuron from sham control (×14,300). B: a degenerating neuronal axon (×3,300). C (×24,475); D (×14,300); E (×42,625); F (×18,150): different stages of apoptotic neurons and glial cells. Symbols: N-Nucleus, M-Mitochondria, R- Rough endoplasmic reticulum, G-Golgi complex, Ch-Chromatin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: TEM confirmation of neuronal and glial apoptosis. The tissue for TEM was obtained at 24 h post-SCI from layer VIII of the ventral horn of the cord and the 2–2.5 mm rostral from the epicenter were processed for TEM as described in the Methods. A: a normal neuron from sham control (×14,300). B: a degenerating neuronal axon (×3,300). C (×24,475); D (×14,300); E (×42,625); F (×18,150): different stages of apoptotic neurons and glial cells. Symbols: N-Nucleus, M-Mitochondria, R- Rough endoplasmic reticulum, G-Golgi complex, Ch-Chromatin.
Mentions: SCI-induced apoptosis was confirmed by TEM identification based on the specific morphology of apoptosis. Figure 5 illustrates the ultra-fine structural changes in apoptotic cells in layer VIII of the ventral horn in the gray matter of the spinal cord sections at 2-2.5 mm rostral from the epicenter removed at 24 h post-SCI (50 g.cm). Figure 5A, a sham control section 2 mm rostral from the epicenter, shows a normal neuron with a large, euchromatic (pale-staining) nucleus (N), abundant rough endoplasmic reticulum (R), mitochondria (M), and Golgi complexes (G) in the cytoplasm. Figure 5B-F show different stages of apoptosis and degeneration from injured animals. B shows a degenerating swollen axon (arrow) with vacuoles. C-F shows very early to late stages of apoptotic degeneration of neuronal and glial cells. C shows a very early stage apoptotic neuron with condensed chromatin (Ch) in the rim of the nucleus (arrow) and unchanged organelle in the cytoplasm. D shows a normal glial cell (arrow) with a smaller cell body and heterochromatic nucleus stained more intensely than the nucleus of the neuron. An apoptotic glial cell near the normal glial cell (arrow head) is shrinking, becoming denser and the chromatin undergoing condensation to form an irregular dense mass while the organelles are becoming indiscernible. E shows the early stage of an apoptotic neuron with condensed chromatin accumulated mainly at the nuclear rim; the organelles are intact. F represents a nucleus which is fragmented and has formed highly condensed apoptotic bodies (arrow head). A phagocyte (arrow) is shown engulfing the apoptotic body.

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