Apoptosis after traumatic human spinal cord injury

Evelyne Emery M.D., Philipp Aldana M.D., Mary Bartlett Bunge Ph.D., William Puckett, Anu Srinivasan Ph.D., Robert W. Keane Ph.D., John Bethea Ph.D., and Allan D. O. Levi M.D., Ph.D., F.R.C.S.(C)
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  • Department of Neurological Surgery and the Miami Project to Cure Paralysis, and Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida; and IDUN Pharmaceuticals, Inc., La Jolla, California
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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 suspected presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1-beta-converting enzyme/Caenorhabditis elegans D 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 CPP-32 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.

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 suspected presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1-beta-converting enzyme/Caenorhabditis elegans D 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 CPP-32 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.

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Contributor Notes

Address reprint requests to: Allan D. O. Levi, M.D., The Miami Project to Cure Paralysis, 1600 NW 10th Avenue, R-48, Miami, Florida 33101. email: alevi@mednet.med.miami.edu.

Address for Dr. Emery: Hôpital Beaujon, Clichy, France.

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