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Neuronal and Glial Apoptosis after Traumatic Spinal Cord Injury

Liu, Xiao Z., Xu, Xiao M., Hu, Rong, Du, Cheng, Zhang, Shu X., McDonald, John W., Dong, Hong X., Wu, Ying J., Fan, Guang S., Jacquin, Mark F., Hsu, Chung Y., Choi, Dennis W.·1997·The Journal of Neuroscience
DOI 10.1523/jneurosci.17-14-05395.1997
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Abstract

Cell death was examined by studying the spinal cords of rats subjected to traumatic insults of mild to moderate severity. Within minutes after mild weight drop impact (a 10 gm weight falling 6.25 mm), neurons in the immediate impact area showed a loss of cytoplasmic Nissl substances. Over the next 7 d, this lesion area expanded and cavitated. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate–biotin nick end labeling (TUNEL)-positive neurons were noted primarily restricted to the gross lesion area 4–24 hr after injury, with a maximum presence at 8 hr after injury. TUNEL-positive glia were present at all stages studied between 4 hr and 14 d, with a maximum presence within the lesion area 24 hr after injury. However 7 d after injury, a second wave of TUNEL-positive glial cells was noted in the white matter peripheral to the lesion and extending at least several millimeters away from the lesion center. The suggestion of apoptosis was supported by electron microscopy, as well as by nuclear staining with Hoechst 33342 dye, and by examination of DNA prepared from the lesion site. Furthermore, repeated intraperitoneal injections of cycloheximide, beginning immediately after a 12.5 mm weight drop insult, produced a substantial reduction in histological evidence of cord damage and in motor dysfunction assessed 4 weeks later. Present data support the hypothesis that apoptosis dependent on active protein synthesis contributes to the neuronal and glial cell death, as well as to the neurological dysfunction, induced by mild-to-moderate severity traumatic insults to the rat spinal cord.

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Figure 1 Schematic drawings showing longitudinal (coronal) sections through the central canal in animals receiving the 6.25 mm impact injury and being perfused at 5 min, 4, 8, and 24 hr, and 3 d ( A ); 7 d ( B ); and 14 and 30 d ( C ). Each contour is the average from four animals. Progressive expansion of the lesion was seen, initially defined by the disappearance of Nissl substance from neurons (5 min-4 hr), later defined by the breaking down of axonal segments and myelin as well as the invasion of blood cells into white matter, and still later defined by gross cavitation (7-30 d, cross-hatched areas ). By 14 d, the lesion consisted entirely of a cavity and was somewhat smaller than the lesion defined at 3 d; this cavity was somewhat increased by 30 d. WM , White matter; GM , gray matter; *Site of impact. Scale bar, 1 mm.
Figure 1 Schematic drawings showing longitudinal (coronal) sections through the central canal in animals receiving the 6.25 mm impact injury and being perfused at 5 min, 4, 8, and 24 hr, and 3 d ( A ); 7 d ( B ); and 14 and 30 d ( C ). Each contour is the average from four animals. Progressive expansion of the lesion was seen, initially defined by the disappearance of Nissl substance from neurons (5 min-4 hr), later defined by the breaking down of axonal segments and myelin as well as the invasion of blood cells into white matter, and still later defined by gross cavitation (7-30 d, cross-hatched areas ). By 14 d, the lesion consisted entirely of a cavity and was somewhat smaller than the lesion defined at 3 d; this cavity was somewhat increased by 30 d. WM , White matter; GM , gray matter; *Site of impact. Scale bar, 1 mm.CC-BY-NC-SA · From the PDF
degenerating axonal segment ( arrowhead ) are also marked for comparison. G -I , Hoechst 33342-stained sections. These show nuclear fragmentation of a probable neuron ( H , arrow ) and a glial cell ( I , arrow ) in the spinal cord 24 hr after injury. The nuclear labeling of a morphologically normal neuron ( G , arrow ) is presented for comparison. WM , White matter; GM , gray matter. Scale bars: A , 50 m m; B -F , 10 m m; G -I , 10 m m.
degenerating axonal segment ( arrowhead ) are also marked for comparison. G -I , Hoechst 33342-stained sections. These show nuclear fragmentation of a probable neuron ( H , arrow ) and a glial cell ( I , arrow ) in the spinal cord 24 hr after injury. The nuclear labeling of a morphologically normal neuron ( G , arrow ) is presented for comparison. WM , White matter; GM , gray matter. Scale bars: A , 50 m m; B -F , 10 m m; G -I , 10 m m.CC-BY-NC-SA · From the PDF
Figure 3 Double staining of cells in the spinal cord after a 6.25 mm injury. TUNEL-positive cells were also positive for neuronal-specific enolase or rip. A , Section from rat euthanized 8 hr after injury showing two shrunken neurons within the lesion area that were labeled simultaneously by TUNEL ( brown ) and neuronal-specific enolase immunohistochemistry ( blue gray ). B , Two other neurons in the same section used in A but outside the lesion area that were labeled for neuronal-specific enolase but were TUNEL negative. C , Section from rat euthanized 7 d after injury showing representative immunofluorescence micrograph showing a spinal cord oligodendrocyte cell body that was located in white matter labeled for TUNEL ( yellow ) as well as rip ( green ). Scale bars: A , B , 10 m m; C , 10 m m.
Figure 3 Double staining of cells in the spinal cord after a 6.25 mm injury. TUNEL-positive cells were also positive for neuronal-specific enolase or rip. A , Section from rat euthanized 8 hr after injury showing two shrunken neurons within the lesion area that were labeled simultaneously by TUNEL ( brown ) and neuronal-specific enolase immunohistochemistry ( blue gray ). B , Two other neurons in the same section used in A but outside the lesion area that were labeled for neuronal-specific enolase but were TUNEL negative. C , Section from rat euthanized 7 d after injury showing representative immunofluorescence micrograph showing a spinal cord oligodendrocyte cell body that was located in white matter labeled for TUNEL ( yellow ) as well as rip ( green ). Scale bars: A , B , 10 m m; C , 10 m m.CC-BY-NC-SA · From the PDF
Image 4
CC-BY-NC-SA · From the PDF
Figure 5 Electron micrograph showing a representative apoptotic cell and a representative necrotic cell in the gray matter of the cord 8 hr after injury. A , Apoptosis. The nucleus has fragmented into several membrane-bounded, highly condensed bodies ( thick arrow ), and the cell body has shrunk with an intact, infolded cell membrane ( arrowheads ). B , Necrosis. The nucleus ( Nu ) is swollen ( arrowhead ) with scattered granular aggregations of chromatin ( thick arrows ), and the cell is swollen with dilation of rough endoplasmic reticulum ( thin arrows ) and mitochondria ( asterisks ). RBC , Red blood cell. Scale bars, 1 m m.
Figure 5 Electron micrograph showing a representative apoptotic cell and a representative necrotic cell in the gray matter of the cord 8 hr after injury. A , Apoptosis. The nucleus has fragmented into several membrane-bounded, highly condensed bodies ( thick arrow ), and the cell body has shrunk with an intact, infolded cell membrane ( arrowheads ). B , Necrosis. The nucleus ( Nu ) is swollen ( arrowhead ) with scattered granular aggregations of chromatin ( thick arrows ), and the cell is swollen with dilation of rough endoplasmic reticulum ( thin arrows ) and mitochondria ( asterisks ). RBC , Red blood cell. Scale bars, 1 m m.CC-BY-NC-SA · From the PDF
Figure 6 Ethidium bromide-stained agarose gel showing a DNA ladder from a rat spinal cord after a weight drop insult. Data are from rats euthanized: in Lane 1 , after no injury (normal control); in lane 2 , 4 hr after injury; in lane 3 , 8 hr after injury; and in lane 4 , 24 hr after injury. Molecular weight markers are shown to the lef t of lane 1 . DNA laddering is apparent in lane 4 ( arrows ).
Figure 6 Ethidium bromide-stained agarose gel showing a DNA ladder from a rat spinal cord after a weight drop insult. Data are from rats euthanized: in Lane 1 , after no injury (normal control); in lane 2 , 4 hr after injury; in lane 3 , 8 hr after injury; and in lane 4 , 24 hr after injury. Molecular weight markers are shown to the lef t of lane 1 . DNA laddering is apparent in lane 4 ( arrows ).CC-BY-NC-SA · From the PDF
Figure 7 Enrichment of mononucleosomes and oligonucleosomes in the cytoplasmic fraction after a 6.25 mm injury ( enrichment factor , absorbance of the injured tissue/absorbance of the normal control tissue). An ELISA kit (see Materials and Methods) was used to detect mononucleosomes and oligonucleosomes in the cytoplasmic fraction of spinal cord tissue at the indicated times after injury. Evidence of internucleosomal DNA fragmentation peaked 24 hr after injury and was reduced by cycloheximide treatment. Error bars indicate SEM; * p , 0.05; n 5 3 animals per group.
Figure 7 Enrichment of mononucleosomes and oligonucleosomes in the cytoplasmic fraction after a 6.25 mm injury ( enrichment factor , absorbance of the injured tissue/absorbance of the normal control tissue). An ELISA kit (see Materials and Methods) was used to detect mononucleosomes and oligonucleosomes in the cytoplasmic fraction of spinal cord tissue at the indicated times after injury. Evidence of internucleosomal DNA fragmentation peaked 24 hr after injury and was reduced by cycloheximide treatment. Error bars indicate SEM; * p , 0.05; n 5 3 animals per group.CC-BY-NC-SA · From the PDF
Figure 8 Hematoxylin- and eosin-stained horizontal cross-sections taken 4 weeks after injury in rats treated with either vehicle ( left ) or cycloheximide ( right ). B , E , Sections through the lesion epicenter (0). A , D , Sections 750 m m rostral to the epicenter ( 2 750 m m). C , F , Sections 750 m m caudal to the epicenter ( 1 750 m m). Scale bar, 100 m m.
Figure 8 Hematoxylin- and eosin-stained horizontal cross-sections taken 4 weeks after injury in rats treated with either vehicle ( left ) or cycloheximide ( right ). B , E , Sections through the lesion epicenter (0). A , D , Sections 750 m m rostral to the epicenter ( 2 750 m m). C , F , Sections 750 m m caudal to the epicenter ( 1 750 m m). Scale bar, 100 m m.CC-BY-NC-SA · From the PDF
Figure 9 Quantitation of histological sparing produced by cycloheximide in animals 4 weeks after a 12.5 mm weight drop insult. The rim of nearly normal tissue (some vacuoles can be seen) was measured in both vehicleand cycloheximide-treated animals. The hypercellular core in cycloheximide-treated animals was considered part of the lesion area and thus not counted as spared tissue. A beneficial effect of cycloheximide on tissue sparing was seen at all levels examined. Error bars indicate SEM; * p , 0.05; n 5 12 animals per group.
Figure 9 Quantitation of histological sparing produced by cycloheximide in animals 4 weeks after a 12.5 mm weight drop insult. The rim of nearly normal tissue (some vacuoles can be seen) was measured in both vehicleand cycloheximide-treated animals. The hypercellular core in cycloheximide-treated animals was considered part of the lesion area and thus not counted as spared tissue. A beneficial effect of cycloheximide on tissue sparing was seen at all levels examined. Error bars indicate SEM; * p , 0.05; n 5 12 animals per group.CC-BY-NC-SA · From the PDF
Figure 10 Hematoxylin- and eosin-stained transverse right hemisection at the lesion epicenter in a rat treated with cycloheximide. A , Section showing a wide rim of spared cord tissue ( arrow ). In the central region, hypercelluar, vascularized tissue with many macrophage-like cells is seen. B , Higher magnification of demarcated region in A showing a border between the peripheral rim of spared cord tissue and this central hypercellular tissue ( dotted line ). BV , Blood vessel. Arrowheads indicate probable macrophages. Scale bars, 100 m m.
Figure 10 Hematoxylin- and eosin-stained transverse right hemisection at the lesion epicenter in a rat treated with cycloheximide. A , Section showing a wide rim of spared cord tissue ( arrow ). In the central region, hypercelluar, vascularized tissue with many macrophage-like cells is seen. B , Higher magnification of demarcated region in A showing a border between the peripheral rim of spared cord tissue and this central hypercellular tissue ( dotted line ). BV , Blood vessel. Arrowheads indicate probable macrophages. Scale bars, 100 m m.CC-BY-NC-SA · From the PDF
Figure 11 Long-term beneficial effect of cycloheximide on the hindlimb neurological function of rats after a 12.5 mm weight drop injury, assessed by the BBB Locomotor Rating Scale. Error bars indicate SEM; * p , 0.05; n 5 12 animals per group.
Figure 11 Long-term beneficial effect of cycloheximide on the hindlimb neurological function of rats after a 12.5 mm weight drop injury, assessed by the BBB Locomotor Rating Scale. Error bars indicate SEM; * p , 0.05; n 5 12 animals per group.CC-BY-NC-SA · From the PDF

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Metadata

Authors
Liu, X. Z., Xu, X. M., Hu, R., Du, C., Zhang, S. X., McDonald, J. W., Dong, H. X., Wu, Y. J., Fan, G. S., Jacquin, M. F., Hsu, C. Y., & Choi, D. W.
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
1997-07-15
Volume
17
Issue
14
Pages
5395-5406
ISSN
  • 0270-6474
  • 1529-2401
DOI
10.1523/jneurosci.17-14-05395.1997
Cited by
770
References
1
Language
en
Licence
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 184 d)

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