Apoptosis after traumatic human spinal cord injury

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Object. Apoptosis is a form of programmed cell death seen in a variety of developmental and disease states, including traumatic injuries. The main objective of this study was to determine whether apoptosis is observed after human spinal cord injury (SCI). The spatial and temporal expression of apoptotic cells as well as the nature of the cells involved in programmed cell death were also investigated.

Methods. The authors examined the spinal cords of 15 patients who died between 3 hours and 2 months after a traumatic SCI. Apoptotic cells were found at the edges of the lesion epicenter and in the adjacent white matter, particularly in the ascending tracts, by using histological (cresyl violet, hematoxylin and eosin) and nuclear staining (Hoechst 33342). The presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1β-converting enzyme/Caenorhabditis elegans D 3 (ICE/CED-3) family of proteases that plays an essential role in programmed cell death. Apoptosis in this series of human SCIs was a prominent pathological finding in 14 of the 15 spinal cords examined when compared with five uninjured control spinal cords. To determine the type of cells undergoing apoptosis, the authors immunostained specimens with a variety of antibodies, including glial fibrillary acidic protein, 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase), and CD45/68. Oligodendrocytes stained with CNPase and a number of apoptotic nuclei colocalized with positive staining for this antibody.

Conclusions. These results support the hypothesis that apoptosis occurs in human SCIs and is accompanied by the activation of caspase-3 of the cysteine protease family. This mechanism of cell death contributes to the secondary injury processes seen after human SCI and may have important clinical implications for the further development of protease inhibitors to prevent programmed cell death.

Article Information

Address for Dr. Emery: Hôpital Beaujon, Clichy, France.Address reprint requests to: Allan D. O. Levi, M.D., The Miami Project to Cure Paralysis, 1600 NW 10th Avenue, R-48, Miami, Florida 33136. email: alevi@mednet.med.miami.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Photomicrographs showing apoptotic nuclei (arrowheads) within the traumatized human spinal cord stained with cresyl violet (A and B), H & E (C), and Hoechst 33342 (D). Apoptotic nuclei appear as condensed, clumped fragments. Bar = 20 µm.

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    Photomicrographs showing TUNEL-labeled cells in the spinal cord at the epicenter level. Cross-sections of the spinal cord demonstrate a single TUNEL-positive labeled nucleus that appears small and fragmented (A and B, arrowhead), among numerous normal nuclei counterstained by propidium iodide nuclear dye (B). On double exposure (C), the apoptotic cell is yellow (arrowhead), whereas the normal nuclei remain red. Bar = 50 µm.

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    Photomicrographs showing CM 1 immunostaining of the dorsal columns at low (A) and high magnification (B) demonstrating a number of dark and shrunken cells (arrows) containing condensed and clumped chromatin. The staining for processed CPP-32 is evident within the nucleus (B). Bar = 200 µm (A), 10 µm (B).

  • View in gallery

    Schematic diagram demonstrating the spatial distribution of apoptotic cells in the spinal cord of the patient in Case 13, who died 17 days postinjury. The apoptotic bodies were identified on cresyl violet—stained sections and are represented as either filled circles at the epicenter level, empty circles above, or filled triangles below the lesion.

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    Photomicrographs showing oligodendrocytes immunostained with CNPase and nuclei counterstained with cresyl violet. Normal oligodendrocytes are seen (A), as well as multiple apoptotic oligodendrocytes (arrows) (B and C). Bar = 50 µm.

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    Photomicrographs showing activated microglia immunostained with CD68 before the section was counterstained with cresyl violet to demonstrate apoptotic bodies. Cells that did not stain with the activated microglia marker (A, arrow) as well as CD68-positive cells ingesting apoptotic bodies (B, arrow) were observed. Bar = 50 µm.

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