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Erkan Kaptanoglu, Hakan Caner, Ihsan Solaroglu and Kamer Kilinc

neuronal death. 9, 27 Apoptosis is regulated by an intracellular proteolytic cascade, mediated by the caspase family. These proteases proteolytically cleave each other and various other target proteins to destroy the cell. 1 Caspase-1 and caspase-3 have been implicated as critical proteases in apoptosis following SCI. 14 Caspase-3 activation has been demonstrated in human neuronal trauma, ischemia, and neurodegenerative diseases. 10, 19, 28 Emery, et al., 7 observed significant labeling for active caspase-3 around SCI sites. It is believed that long

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Dali Yin, Norihiko Tamaki and Takashi Kokunai

subjected to electrophoresis on a 2% agarose gel for analysis of amplification results. A PCR reaction in which primers for GAPDH were used was performed as an internal control. Caspase-3 and ICE Colorimetric Assay Among members of the ICE family, ICE and CPP32 have different substrate specificities. We examined the involvement of ICE and CPP32 in etoposide-induced apoptosis. Caspase assay kits provide a simple and convenient means for assaying CPP32 and ICE protease activity. Briefly, harvested cells (2 ×10 6 ) were centrifuged and washed twice with cold PBS. The cell

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Evelyne Emery, Philipp Aldana, Mary Bartlett Bunge, William Puckett, Anu Srinivasan, Robert W. Keane, John Bethea and Allan D. O. Levi

leads to membrane lysis and release of intracellular constituents that evoke an inflammatory reaction. 10, 37, 43 Apoptosis has long been known to occur as a form of neuronal cell death during embryonic development 22, 25 and has been observed more recently following damage to the nervous system caused by ischemia, neurodegenerative conditions, inflammatory diseases, and traumatic injuries. 3, 6, 22, 24, 41, 44, 46, 50, 54 Caspases are a family of cysteine proteases that play an important role in the effector phase of apoptosis. Caspase-3, in particular, has been

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Evelyne Emery, Philipp Aldana, Mary Bartlett Bunge, William Puckett, Anu Srinivasan, Robert W. Keane, John Bethea and Allan D. O. Levi

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.

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-1-beta-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.

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.

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Evelyne Emery, Philipp Aldana, Mary Bartlett Bunge, William Puckett, Anu Srinivasan, Robert W. Keane, John Bethea and Allan D. O. Levi

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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Narendra Nathoo, Pradeep K. Narotam, Devendra K. Agrawal, Catherine A. Connolly, James R. van Dellen, Gene H. Barnett and Runjan Chetty

to evaluate the status of the apoptosis-related cell proteins p53, bax, bcl-2, and caspase-3 in the PIZ. To determine the presence of apoptosis and apoptosis-related proteins in control (non-TBI) brain tissue, samples were harvested in the same volume from “normal” margins of resection in three patients who were undergoing surgery for temporal lobe epilepsy; these samples were then processed in a manner identical to that used for the PIZ tissue. Immunohistochemical Findings Apoptosis-Related Proteins bax, bcl-2, and p53. Two-micrometer-thick sections of wax

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Hongtao Wu, Dunyue Lu, Hao Jiang, Ye Xiong, Changsheng Qu, Bo Li, Asim Mahmood, Dong Zhou and Michael Chopp

different areas, 10 consecutive fields were randomly selected and evaluated at 400 × using an imaging analyzer. A blind-counting method was used to count TUNEL-positive cell numbers. For counting, images were obtained from the cortex, hippocampus, and dentate gyrus of the ipsilateral side of the injured animals. In each image we manually counted the TUNEL-positive cells from the boundary zone in the cortex, the CA3 region in the hippocampus, and the body and hilus of the dentate gyrus. Caspase-3 Activity Assay The activity of caspase-3 was determined using an Apo

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Chih-Lung Lin, Aaron S. Dumont, Yu-Feng Su, Zen-Kong Dai, Juei-Tang Cheng, Yee-Jean Tsai, Jih-Hui Huang, Kao-Ping Chang and Shiuh-Lin Hwang

SAH. 17 , 29 To date, apoptosis has been studied extensively in stroke and to a more limited degree in SAH. In the present study, we examined potential antiapoptotic effects of E2. We specifically attempted to clarify the following points: 1) that SAH leads to cell death in the rat brain; 2) that the apoptotic pathway contributes to brain injury after SAH; 3) that E2 acts on specific apoptotic factors including TNF-α, caspase 3, Bcl-2, and Bax to prevent SAH-induced apoptotic death; and 4) that E2 has antiapoptotic effects against brain injury after SAH via ER

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Matthew Wetzel, Daniel R. D. Premkumar, Beth Arnold and Ian F. Pollack

% bovine serum albumin in Tris-buffered saline and 0.1% Tween 20 at room temperature for 1 hour and probed with primary antibodies against acetyl-histone H2B (Lys12), acetyl-histone H3 (Lys18), acetyl-histone H4 (Lys12), PARP, p21 WAF1 , phosphorylated Rb (Ser807/811), and caspase-3 (all from Cell Signaling Technology, Inc., Beverly, MA) for 16 hours at 4°C. After washing, the membranes were incubated with species-specific horseradish peroxidase—conjugated secondary antibodies (mouse and rabbit; Cell Signaling Technology) in Tris-buffered saline—Tween 20 at room

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Haitao Ju, Xin Li, Hong Li, Xiaojuan Wang, Hongwei Wang, Yang Li, Changwu Dou and Gang Zhao

electrophoresis, and the bands were quantified with ImageQuant, Version 5.0 software (Molecular Dynamics, Inc.). Antibodies and Western Blotting Anti-STAT1, anti-p21, anti-PCNA, anti-Bax, anti–caspase-9, anti–Bcl-2, anti–β-actin, and anti–caspase-3 antibodies were obtained from Santa Cruz Biotechnology, Inc. For Western blot analyses, cells were harvested at 48 hours after transfection and lysed with lysis buffer (TaKaRa Bio, Inc.). After processing in a centrifuge at 15,000 g for 30 minutes, the supernatants were analyzed for protein content using the Bradford reagent