Apoptosis after traumatic human spinal cord injury

Full access

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.

Abstract

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.

Apoptosis is an important biological process in eukaryotes in which individual cells die by activating an intrinsic suicide mechanism. Apoptosis is distinguished from necrotic cell death by morphological and biochemical criteria. Apoptosis is an active process of cell destruction characterized by cell shrinkage, chromatin aggregation with genomic fragmentation, and nuclear pyknosis.27,31 In contrast, necrosis is characterized by passive cell swelling, intense mitochondrial damage with rapid energy loss, and disruption of internal homeostasis. Necrosis 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 development22,25 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 shown to be important in neural development and injury. Germline deletions of this protease led to severe neurological defects in mice.34 The contribution of caspace-3 activity and apoptosis to neuronal cell death after traumatic brain injury54 and experimental transient ischemia24,41 has been reported. In both injury paradigms, the use of caspase inhibitors not only reduced the extent of apoptosis, but also resulted in functional behavioral improvement in the animals.

The presence of apoptosis in spinal cord injury (SCI) following a contusion has been reported recently in rats and monkeys.11,32,35,36 In these studies, it was shown that apoptosis contributed to the tissue damage seen after SCI. Apoptotic cell death was observed in both neurons and oligodendrocytes11,36 and was prominent in the white matter, in which wallerian degeneration was simultaneously observed. A time course analysis in rats36 revealed that apoptosis occurred as early as 4 hours postinjury and could be seen in decreasing amounts as late as 3 weeks after SCI.

We initiated this study to determine whether apoptosis is an important factor after human SCI by examining the spinal cords of patients who died between 3 hours and 2 months postinjury. Apoptosis in these 15 patients was assessed using multiple criteria, including nuclear morphology, chromatin staining techniques, and immunostaining for a processed form of caspase-3. We also determined the spatial and temporal expression of apoptotic cells and the nature of the cells involved in programmed cell death.

Clinical Material and Methods
Patient Population

This study was based on postmortem examination of spinal cord tissue from five control patients without SCI and 15 patients who died at different time points after an SCI. Fifteen specimens were obtained from the Miami Project's Human Spinal Cord Injury Bank, which contains more than 100 injured human spinal cords. The cases were selected based on the short interval between SCI and death (0–2 months) and the availability of tissue above and below the lesion site.

Preparation of Specimens

The spinal cords had been removed at autopsy, usually within 16 hours after death. They were then fixed in 10% neutral buffered formalin for a minimum of 15 days, after which they were stored in 0.1 M phosphate buffer at 4°C. Selected tissue blocks were taken at the epicenter and at three consecutive root levels above and below the injury. Samples from each of these regions were dehydrated, embedded in paraffin, and cut in 7-µm-thick cross-sections.

Sections were stained with hematoxylin and eosin and cresyl violet (15 samples), terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridinetriphosphate nick-end labeling ([TUNEL], five samples), or Hoechst 33342 (four samples), and immunostained with CM 1, an antibody that preferentially recognizes the processed form of caspase-3 (10 samples). Sections from control spinal cords (five samples) were stained with cresyl violet and immunostained with the CM 1 antibody. Wallerian degeneration in white matter tracts was assessed using a silver stain for axons (Sevier—Munger) and a myelin stain (solochrome—cyanine) on adjacent sections. Regions undergoing wallerian degeneration contained swollen, fragmented, or absent axons in the same areas in which myelin was distorted or collapsed. Wallerian degeneration was quantified in the white matter above and below the lesion according to four levels of severity: subtle, mild, moderate, and severe.

Staining With the TUNEL Technique

To determine whether DNA fragmentation characteristic of apoptosis occurs, we stained spinal cord tissue by using the in situ TUNEL technique.19 Spinal cord sections were deparaffinized in xylene for 5 minutes and then rehydrated sequentially in 100%, 95%, 75%, and 50% ethanol. The sections were incubated with 50 µg/ml proteinase K for 5 minutes to strip off nuclear proteins. The TUNEL staining was completed using the apoptosis in situ kit according to the manufacturer's instructions.

Briefly, sections were immersed in equilibration buffer for 10 minutes and then incubated with TdT and deoxyuridinetriphosphate-fluorescein isothiocyanate in a humidified chamber at 37°C for 1 hour. The sections were washed with 0.1 M phosphate-buffered saline (PBS) at pH 7.4 and counterstained with propidium iodide (a nuclear stain) for 10 minutes. As a positive control we pretreated slides with DNAase to produce TUNEL-positive staining of all nuclei, and the negative controls were incubated without TdT enzyme. Sections were examined and photographed using fluorescence microscopy.

Hoechst 33342 Staining

Spinal cord sections were deparaffinized with xylene for 5 minutes, rehydrated, and rinsed with 0.1 M PBS. The sections were stained with one drop of glycerol/PBS solution containing 5 µl Hoechst 33342 dye (5.6 mg in 10 ml PBS).

Immunostaining for Processed Form of Caspase-3 With the CM 1 Antibody

Spinal cord sections were deparaffinized as described earlier, rinsed in 0.01 M PBS, and treated for 30 minutes with 3% H2O2 in 10% methanol. The sections were blocked for 1 hour in 5% normal goat serum with 0.1 M PBS and 0.1% Triton X-100. The primary antibody CM 1 (0.012 µg/ml) was applied and the preparations were left overnight. The following morning, the sections were fixed for 10 minutes in 0.01 M PBS containing 0.1% glutaraldehyde. The sections were incubated for 1 hour with a biotinylated secondary antibody, followed by a 1-hour incubation with an avidin—biotin complex (ABC) reagent. Sections were then incubated for 10 minutes in 0.5% biotinylated tyramine 1% H2O2 solution in 0.01 M PBS. They were reincubated1 in the ABC reagent for 30 minutes. The stain was developed in diaminobenzidine and H2O2 and enhanced by nickel chloride in PBS. The sections were rinsed, dehydrated, mounted on a coverslip, and examined under the light microscope. Sections from uninjured control cases were processed using the same protocol.

Immunostaining for Glial Fibrillary Acidic Protein, CNPase, CD45, and CD68

Immunohistochemical techniques were used to detect the following antigens: glial fibrillary acidic protein (GFAP), 2′, 3′-cyclic nucleotide 3′-phosphohydrolase (CNPase), and CD45/CD68. Glial fibrillary acidic protein GFAP is an astrocyte marker; CNPase is a myelin protein in the central nervous system and has been shown to be specific for oligodendrocytes;45 and CD45 and CD68 were used to study activated microglia and macrophages.

Tissue sections were deparaffinized and endogenous peroxidase activity was blocked with 0.3% H2O2 in 100% methanol. Sections were incubated with either 0.3% Triton X-100 in 0.1 M PBS (GFAP) or with Tris (pH 7.6) containing 10% normal goat serum. The sections were incubated overnight at room temperature with the following monoclonal antibodies: GFAP 1:200, CD45 1:100, CD68 1:100, and CNPase 1:800. This was followed by sequential incubation with biotinylated secondary antibody, ABC with horseradish peroxidase, 3,3′-diaminobenzidine, and H2O2. The slides were then counterstained with 2% cresyl violet, dehydrated, and mounted. Omission of the primary antibody served as a negative control.

Sources of Supplies and Equipment

The proteinase K and Hoechst 33342 dye were purchased from Sigma Chemical Co., St. Louis, MO. The apoptosis in situ kit was acquired from Chemicon International, Inc., Temecula, CA. The Axiophot fluorescent microscope was obtained from Zeiss, Inc., Oberkochen, Germany. The primary antibody CM 1 was a generous gift from IDUN Pharmaceuticals, Inc., La Jolla, CA. The biotinylated secondary antibody and ABC reagent (Vectastain) were acquired from Vector Laboratories, Burlingame, CA. The monoclonal antibodies GFAP, CD45, and CD68 were purchased from Dako Corp., Carpenteria, CA, and the CNPase from Sternberger Monoclonals, Inc., Baltimore, MD.

Results
Clinical Data

The mean age of the 15 patients with SCIs (Table 1) was 49 years (range 16–80 years) and the mean age of the five control patients was 55 years (range 33–77 years). The principal causes of death in the patients with SCIs were pulmonary embolism (six patients), pneumonia (six patients), hemothorax with cardiac arrest in one, and an unknown cause in two patients. The causes of death for the control patients were cardiovascular disease in two and a motor vehicle accident (without head injury or SCI) in three. Only two patients with SCI (Cases 4 and 5) received methylprednisolone according to the Second National Acute Spinal Cord Injury Study protocol.8

TABLE 1

Summary of the clinical data of the 15 patients with SCI*

Case No.Survival TimeAge (yrs), SexInjury LevelCause of InjuryNeurological StatusClassification of InjuryHistopathological Findings at Lesion EpicenterPresence & Location of Wallerian Degeneration
13 hrs 72, MT-6fallcompletemassive compressionmaceration & hemorrhagic componentsno wallerian degeneration
23 days 67, MC-6fallincompletesolid cordminor increase in interstitial spacessubtle, above
36 days 74, MC6–7MVAcompletemassive compressionall neural tissue damaged at epicentermild, above
48 days 20, FC7–8GSWcompletelacerationsubtotal destruction of neural tissue at epicentermoderate, above & below
510 days 22, MC-7divingincompletecontusionmacerated tissue & phagocytic response atmild, asymmetric; above &
  epicenter below
612 days 59, MC-6MVAcompletecontusionmajor loss of neural tissue at epicentersevere, above & below
712 days 16, FT-11GSWcompletelacerationall neural tissue damaged at epicentervery subtle, above & below
812 days 19, ML-1GSWcompletecontusionmacerated tissue at epicenter; vascular infarctionsevere, up to C-3 & below
  proximal to epicenter
915 days 38, MT-5GSWcompletelacerationspinal cord transected at T-5; necrotic tissue at T-4moderate, above & below
  w/ invasion of macrophages
1016 days 43, MC5–6fallcompletecontusionlarge blood-filled cavities w/ macrophage invasionmild
  at epicenter; preserved axon/myelin units in
  extreme periphery of most of cord
1116 days 80, MC-6fallcompletecontusionlarge destruction of cord tissue w/ macrophages &mild, more present above
  hemorrhage than below
1217 days 70, MC-8MVAcompletecontusionloss of neural tissue at epicentermoderate, above & below
1317 days 17, FC6–7MVAcompletecontusionmacerated tissue in ventral & central portions ofmoderate, more present
  cord at epicenter above than below
1445 days 67, MC5–7fallcompletecontusionlarge destruction of cord tissue at epicentermoderate, above & below
1560 days 68, FT-5fallincompletecontusionloss of motor neurons at epicenter & diffuse axonalsevere in dorsal columns
  injury throughout dorsal columns & lateral &
  ventral corticospinal tracts

GSW = gunshot wound; MVA = motor vehicle accident.

Received methylprednisolone according to the Second National Acute Spinal Cord Injury Study protocol.

Ten patients suffered a cervical and four a thoracic SCI, and one case consisted of a conus medullaris injury. Twelve patients presented with a complete motor and sensory loss below the level of their injury, whereas three experienced incomplete loss. The cause of injury was variable and included falls (six patients), motor vehicle accidents (four patients), gunshot wounds (four patients), and a diving accident (one patient).

Histological Data

We analyzed cross-sections of the lesion epicenter and rostral and caudal levels in tissue obtained in 15 patients who died between 3 hours and 60 days after their SCI. According to the classification established by Bunge, et al.,9 nine patients presented with a contusion/cavity lesion, two with a massive compression, three with a laceration lesion, and one with a solid spinal cord syndrome.

All patients except the one in Case 2 showed significant destruction of the spinal cord parenchyma at the epicenter of the injury. Signs of wallerian degeneration in the white matter occurred as early as Day 3 postinjury and thereafter spread rostral and caudal to the lesion, with more advanced degeneration of the ascending tracts. In one patient (Case 8), the contusion was associated with a vascular infarction proximal to the epicenter and a pattern of obvious wallerian degeneration.

Morphological Appearance of Apoptotic Cells

Apoptotic nuclei were found in 14 of the 15 cases on hematoxylin and eosin— and cresyl violet—stained sections (Fig. 1A–C). Cells containing apoptotic nuclei exhibited evidence of cytoplasmic shrinkage. The nuclei themselves demonstrated chromatin condensation or aggregation into small and dark nuclear fragments. Staining with Hoechst 33342 revealed typically small and bright fragmented nuclei (Fig. 1D). No apoptotic bodies were detected on cresyl violet—stained sections from the five uninjured control patients.

Fig. 1.
Fig. 1.

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.

The in situ TUNEL technique (Fig. 2A) combined with propidium iodide (Fig. 2B) also demonstrated apoptotic TUNEL-positive nuclei as double-stained (yellow), fragmented nuclei (Fig. 2C) among numerous propidium iodide-positive cells. The location and quantity of apoptotic nuclei observed using this technique correlated with the number of apoptotic nuclei observed with hematoxylin and eosin— and cresyl violet—stained sections (paired t-test, p = 0.16; correlation coefficient 0.83).

Fig. 2.
Fig. 2.

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.

The presence of apoptotic cells was also demonstrated in sections immunostained with the CM 1 antibody. Most positive nuclei were dark and shrunken and contained condensed, clumped chromatin (Fig. 3A and B). Relatively more apoptotic cells were detected after CM 1 antibody staining compared with cresyl violet staining. As cresyl violet staining was used to detect apoptotic bodies and caspase-3—positive cells did not necessarily contain these bodies, the implication is that immunostaining for caspase-3 activation detects apoptosis at an earlier stage. Staining with caspace-3 was also negative in Case 10, in which no apoptotic cells were shown in cresyl violet—stained sections. Staining with caspase-3 showed one to two positive cells per section in tissue obtained in one control patient (73 years old, died of cardiovascular disease), whereas tissue from the four other uninjured control patients was negative. Other potential explanations for the few apoptotic cells seen in tissue obtained in the one control case are the patient's advanced age and associated vascular disease.

Fig. 3.
Fig. 3.

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).

Temporal and Spatial Distribution of Apoptotic Cells

Apoptotic cells were seen between 3 hours and 2 months postinjury. Although too few cases were analyzed to allow a correlation between the time after SCI and the number of apoptotic cells, most of these cells were seen in tissue obtained in patients who died between 3 days and 3 weeks after the SCI. Incomplete neurological injury was associated with fewer apoptotic bodies (Cases 2, 5, and 15). When ischemia was associated with the primary injury, as in Case 8, a large number of apoptotic nuclei were observed throughout the levels studied.

Most apoptotic cells were randomly distributed in the rim of surviving tissue around the epicenter of the SCI and within the adjacent white matter (Table 2), and none was identified in the gray matter. Apoptotic cells were seen in areas of wallerian degeneration in the white matter above and below the epicenter. Rostral to the lesion epicenter, apoptosis was associated with axonal degeneration in ascending tracts, especially the funiculus gracilis, the spinoreticular, the spinothalamic, and the spinocerebellar tracts. Degeneration and apoptosis were absent in the regions containing descending motor pathways. Apoptosis was associated with the degenerating axons in the descending tracts caudal to the lesion, especially the ventral corticospinal, reticulospinal, and vestibulospinal tracts; it was less prevalent in the lateral corticospinal or rubrospinal, and no apoptotic cells were seen in the caudal ascending tracts. Apoptotic cells were seen in much larger numbers in ascending than in descending tracts, and wallerian degeneration was present earlier in the ascending tracts than in the descending tracts. There appeared to be a good correlation between apoptosis and wallerian degeneration. A schematic diagram (Fig. 4) demonstrates the spatial distribution of apoptotic cells at the SCI epicenter as well as above and below the lesion in the spinal cord of the patient in Case 13, who died 17 days postinjury.

TABLE 2

Distribution and quantification of apoptotic cells per section

Case No.Injury LevelApoptotic CellsEpicenter Level1–3 Levels Above Epicenter1–3 Levels Below Epicenter
1T-6yesno viable tissue normal tissue* T-5; no apoptotic cells above T-5 noraml tissue; no apoptotic cells 
2C-6yesedges of lesion* dorsal columns* ventral CST, lat CST*; no apoptotic cells below C-8 
3C6–7yesno viable tissue dorsal columns** at edges of central cavity* 
4C7–8yesedges of lesion** dorsal columns (fx gracilis)* ventral CST (T-1)*; no apoptotic cells below T-2 
5C-7yesedges of cavity* no apoptotic cells from C5–7 dispersed at C-8*; no apoptotic cells at T-1 & below 
6C-6yesedges of lesion* dorsal columns, spinocerebellar tracts** ventral CST* 
7T-11yesedges of lesion* dorsal columns* dispersed T12—L1*; no apoptotic cells below L-2 
8L-1yesno viable tissue dorsal columns (T-11) & a few cells in CST & ventral CST L3—5** 
 spinothalamic tracts***  
9T-5yesno viable tissue spinothalamic tract (T-3)* no apoptotic cells T4–8 
10C5–6no   
11C-6yesedges of lesion** dorsal columns (fx gracilis); spinothalamic tracts* ventral CST** 
12C-8yesedges of lesion** dorsal columns & spinocerebellar tracts** no apoptotic cells at T-1 & below 
13C6–7yesedges of lesion*** dorsal columns & spinothalamic tracts** ventral CST & lat CST** 
14C5–7yesedges of lesion* dorsal columns* ventral CST & lat CST 
15T-5yesedges of lesion* dorsal columns*; no apoptotic cells above T-3 vetral CST* 

Asterisks represent the stratification of cell counts per section: * = 1–15; ** = 15–30; *** = 30–45. Abbreviations: CST = corticospinal tract; fx = funiculus; — = not applicable

Fig. 4.
Fig. 4.

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.

Apoptotic Cell Type

To identify the type of cells undergoing apoptosis, different antibodies directed against astrocytes (GFAP), oligodendrocytes (CNPase), and macrophages or activated microglia markers (CD45, CD68) were assayed. Apoptotic bodies were not seen in astrocytes (data not shown). Immunostaining with CNPase demonstrated that apoptotic cells (identified using cresyl violet counterstain) were present within oligodendrocytes adjacent to myelin sheaths in degenerating white matter tracts (Fig. 5). Macrophages or activated microglia were seen engulfing fragments of apoptotic cells. Apoptotic bodies in proximity to cells unstained with microglia markers were also observed (Fig. 6).

Fig. 5.
Fig. 5.

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.

Fig. 6.
Fig. 6.

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.

Discussion

Apoptosis is a form of physiological cell death, also defined as programmed cell death, in which cells die and are engulfed by phagocytes without discharging cytosolic contents into the extracellular space and without initiating an inflammatory reaction.10 The cell surface membrane begins to form blebs and express prophagocytic signals, the cell shrinks and severs contact with its neighbors, chromatin becomes condensed and cleared, and eventually the whole cell fragments into membrane-bound vesicles that are rapidly ingested by neighboring cells. The apoptotic process can be rapid when compared with necrotic cell death, and the debris is removed with similar swiftness.27,37 Because the process is rapid, quantification of the number of apoptotic cells on any given cross-section underestimates the extent to which apoptosis contributes to the death of cells at the injury site.52

The genetic control and the biochemical markers of apoptotic cell death were initially elucidated in the roundworm Caenorhabditis elegans, and CED 3 was identified as a gene encoding a protein involved in programmed cell death in this maturing roundworm.29 The mammalian homologs of cell death mechanisms consist of the CED 3/ICE (interleukin-1β-converting enzyme) family of cysteine proteases (caspases),17,38,40 in which the prototype is the ICE. The mammalian caspase family is composed of at least 10 known members.2 One of these is caspase-3 (Yama, apopain), which has been definitively implicated in apoptosis.16,26 The essential role of caspases in vertebrate apoptosis is consistent with their activity as the principal effectors of apoptosis through their proteolytic action on specific targets. One of the final effectors of cell death is activation of endonucleases that induce fragmentation of nuclear DNA into 185-bp fragments.30 In a number of reports caspases have been implicated as important during apoptosis of neurons and astrocytes, and it has been suggested that caspase-3 was the principal effector in the apoptotic pathway.4,33 Moreover, caspase-3 activation was never observed in necrotic cell death.4,26 Hisahara, et al.,26 demonstrated that caspase-1 (ICE) and caspase-3 (CPP-32) were expressed in oligodendrocytes and that their inhibition prevented apoptotic cell death.

Secondary injury processes are believed to be an important, remediable component of SCI.8,49 Many of the basic research advances that have reached the clinical arena focus on the prevention of these secondary injury mechanisms.21,55 Apoptotic cell death has been recognized for many years; however, this process has now gained increasing attention in the basic science literature as a mechanism by which cells die in a number of neurological diseases.47 Apoptotic cell death appears to be yet another mechanism in which cells may die in a delayed fashion after injury, that is, a secondary injury mechanism.

There is evidence that apoptotic cell death contributes to tissue damage, and prevention of this process results in neurological recovery after SCI36 and brain injury in rats.54 Data presented here indicate for the first time that apoptosis is associated with the tissue damage observed after human SCI. Our determination of apoptosis relied on multiple criteria: morphological staining (cresyl violet, hematoxylin and eosin), nuclear chromatin staining with Hoechst 33342 dye, and the TUNEL test, all of which have been widely used for assessment of apoptosis. These results were confirmed using immunostaining with the CM 1 antibody that is specific for the processed form of caspase-3 (A Srinivasan, et al., unpublished data). Caspase-3 is required for DNA fragmentation and the morphological changes associated with apoptosis.30

Crowe, et al.,12 presented the first evidence for the presence of apoptosis in SCIs in the rat. Li, et al.,35 demonstrated that compression trauma to the spinal cord was associated with apoptosis of glial cells preferentially located in degenerating longitudinal tracts of the white matter. The apoptotic cells were most likely oligodendrocytes, a conclusion based on morphological data and negative GFAP staining. Further studies demonstrated the occurrence of apoptosis in SCI in rats and monkeys and showed that oligodendrocytes were the major cell population undergoing apoptosis based on immunohistochemical analysis.11,35 Apoptosis of oligodendrocytes was seen in areas of wallerian degeneration and was detected from 24 hours to 3 weeks postinjury.11,12 Liu, et al.,36 also observed a burst of neuronal and glial apoptosis in gray and white matter at the lesion site within the first 24 hours postinjury and a delayed wave of oligodendrocyte apoptosis in distant white matter several days later.

In our study, the lesion epicenter demonstrated the presence of multifocal hemorrhages and necrotic tissue involving the central gray matter and the contiguous white matter. Apoptotic cells were identified surrounding the lesion epicenter. As early wallerian degeneration occurred in the white matter tracts, a second phase of apoptotic cells appeared and these were positive for an oligodendrocyte marker (CNPase). Oligodendrocyte apoptosis was clearly associated with wallerian degeneration and was more obvious in ascending than in descending tracts. This corresponds to the pattern of progression of wallerian degeneration in which the ascending tracts show signs of degeneration before the descending tracts.9

We found no relationship between the average number of apoptotic bodies and time from injury because the injury mechanism and severity were different in each case. Nonetheless, it seemed that apoptosis was less severe in patients with incomplete neurological injuries. Liu, et al.,36 demonstrated that after rat SCI, apoptotic glial cells were more abundant above than below the site of compression and that apoptotic cells were more numerous after moderate and severe injury compared with mild compression. We found no evidence of apoptosis within the spinal cord neurons. This indicates that any neuronal loss was the result of necrosis rather than apoptosis or that it occurred at an early stage before we could detect it. Tissue in only one of our cases was examined within 24 hours of death. Li, et al.,35 demonstrated that neuronal apoptosis was complete within the first 24 hours, whereas Liu and colleagues36 saw no evidence of apoptosis in the spinal cord neurons at 4 hours or 1, 4, and 9 days after compression trauma. We suspect that activated microglia (macrophages) clear apoptotic bodies, as has been observed by others,13,14,48,51 but may also be partly responsible for the induction of apoptosis by secreting cytotoxic substances such as cytokines (tumor necrosis factor—α) and nitric oxide.53 Other investigators have also demonstrated that microglia can undergo apoptosis following damage to the nervous system.20,42

The mechanisms responsible for oligodendrocyte apoptosis remain unclear. It may occur as a result of loss of axonally derived survival signals (wallerian degeneration)5 and/or as a result of evolving adverse changes in the cellular milieu13 resulting in axonal demyelination. If these axons are in continuity across the injury site, electrical conduction will be impaired through the axon.The presence of demyelinated axons around the epicenter of SCIs has received attention recently.7,8 One therapeutic strategy for SCIs relies on the use of the drug 4-amino pyridine (a potassium channel blocker) to enhance axonal conduction through areas of demyelination.23 Thus, prevention of axonal demyelination after SCI by the reduction of apoptotic oligodendrocyte cell death may result in an overall reduction of partially injured axons.

The contribution of CPP-32 activity and apoptosis to neuronal cell death after traumatic brain injury54 and experimental transient ischemia24,41 has been reported. In both injury paradigms, the use of caspase inhibitors not only reduced the extent of apoptosis, but also resulted in functional behavioral improvement in the animals. In that regard, therapeutic interventions aimed at blocking apoptosis may be useful in reducing tissue damage after SCI and ultimately in improving functional outcomes. Liu, et al.,36 reported that intraperitoneal injections of cycloheximide, a protein synthesis inhibitor, improved behavioral outcomes after spinal cord contusion injury in rats. Partial inhibition of protein synthesis can induce the production of Bcl-2,28 an antiapoptotic human protooncogene important in cell survival that was shown to be upregulated in injured axons of the white matter following compression injury of the spinal cord.18,35 Hisahara, et al.,26 demonstrated that caspases were involved in tumor necrosis factor-mediated cell death of oligodendrocytes and that inhibition of these proteases can prevent apoptosis. Milligan, et al.,39 identified peptide inhibitors of the ICE protease family that arrest programmed cell death of motor neurons in vivo and in vitro. In that regard, it would be useful to test drugs that inhibit caspases in the treatment of SCI.

Conclusions

This work demonstrates for the first time that apoptotic cell death is observed from 3 hours to 8 weeks after traumatic human SCIs. Apoptosis occurs around the lesion epicenter as well as within areas of wallerian degeneration in both ascending and descending white matter tracts. Oligodendrocytes were definitely implicated as cells undergoing apoptosis on sections of injured spinal cord in which immunohistochemical markers were used. Apoptosis after human SCI appears to be dependent on activation of CPP-32. Inhibition of this process may have potential therapeutic benefits for reducing tissue damage and improving the outcome after SCI.

Acknowledgments

We are grateful to Tesha Monteith for her technical assistance and to Dr. Alexander Marcillo for his assistance in preparing the manuscript. We also thank Drs. Jacqueline Bresnahan and Hans Lassmann for their helpful advice.

References

  • 1.

    Adams JC: Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40:145714631992Adams JC: Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40:

  • 2.

    Alnemri ESLivingston DJNicholson DWet al: Human ICE/CED-3 protease nomenclature. Cell 87:1711996 (Letter)Cell 87:

  • 3.

    Arends MJWyllie AH: Apoptosis: mechanisms and roles in pathology. Int Rev Exp Pathol 32:2232541991Int Rev Exp Pathol 32:

  • 4.

    Armstrong RCAja TJHoang KDet al: Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis. J Neurosci 17:5535621997J Neurosci 17:

  • 5.

    Barres BAJacobson MDSchmid R: Does oligodendrocyte survival depend on axons? Curr Biol 3:4894971993Curr Biol 3:

  • 6.

    Bauer JWekerle HLassmann H: Apoptosis in brain-specific autoimmune disease. Curr Opin Immunol 7:8398431995Curr Opin Immunol 7:

  • 7.

    Blight AR: Effect of 4-aminopyridine on axonal conduction-block in chronic spinal cord injury. Brain Res Bull 22:47521989Blight AR: Effect of 4-aminopyridine on axonal conduction-block in chronic spinal cord injury. Brain Res Bull 22:

  • 8.

    Bracken MBShepard MJCollins WFet al: A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the second National Acute Spinal Cord Injury Study. N Engl J Med 322:140514111990N Engl J Med 322:

  • 9.

    Bunge RPPuckett WRBecerra JLet al: Observations on the pathology of human spinal cord injury. A review and classification of 22 new cases with details from a case of chronic cord compression with extensive focal demyelination. Adv Neurol 59:75891993Adv Neurol 59:

  • 10.

    Cohen JJ: Apoptosis. Immunol Today 14:1261301993Cohen JJ: Apoptosis. Immunol Today 14:

  • 11.

    Crowe MJBresnahan JCShuman SL: Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nature Med 3:73761997Nature Med 3:

  • 12.

    Crowe MJShuman SLMasters JNet al: Morphological evidence suggesting apoptotic nuclei in spinal cord injury. Soc Neurosci Abstr 21:2321995 (Abstract)Soc Neurosci Abstr 21:

  • 13.

    Dusart ISchwab ME: Secondary cell death and the inflammatory reaction after dorsal hemisection of the rat spinal cord. Eur J Neurosci 6:7127241994Eur J Neurosci 6:

  • 14.

    Duvall EWyllie AHMorris RG: Macrophage recognition of cells undergoing programmed cell death (apoptosis). Immunology 56:3513581985Immunology 56:

  • 15.

    Ellis REYvan JHorvitz HR: Mechanisms and functions of cell death. Annu Rev Cell Biol 7:6636981991Annu Rev Cell Biol 7:

  • 16.

    Enari MTalanian RVWong WWet al: Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature 380:7237261996 (Letter)Nature 380:

  • 17.

    Fraser AEvan G: A license to kill. Cell 85:7817841996Cell 85:

  • 18.

    Furukawa KEstus SFu WMet al: Neuroprotective action of cycloheximide involves induction of Bcl-2 and antioxidant pathways. J Cell Biol 136:113711491997J Cell Biol 136:

  • 19.

    Gavrieli YSherman YBen-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:4935011992in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:

  • 20.

    Gehrmann JBanati RB: Microglial turnover in the injured CNS: activated microglia undergo delayed DNA fragmentation following peripheral nerve injury. J Neuropathol Exp Neurol 54:6806881995J Neuropathol Exp Neurol 54:

  • 21.

    Hall EDYonkers PAAndrus PKet al: Biochemistry and pharmacology of lipid antioxidants in acute brain and spinal cord injury. J Neurotrauma 9 (Suppl):S425S4421992J Neurotrauma 9 (Suppl):

  • 22.

    Hamburger V: Cell death in the development of the lateral motor column of the chick embryo. J Comp Neurol 160:5355461975Hamburger V: Cell death in the development of the lateral motor column of the chick embryo. J Comp Neurol 160:

  • 23.

    Hansebout RRBlight ARFawcett Set al: 4-Aminopyridine in chronic spinal cord injury: a controlled, double-blind, crossover study in eight patients. J Neurotrauma 10:1181993J Neurotrauma 10:

  • 24.

    Hara HFriedlander RMGagliardini Vet al: Inhibition of interleukin 1β converting enzyme family proteases reduce ischemic and excitotoxic neuronal damage. Proc Natl Acad Sci USA 94:200720121997Proc Natl Acad Sci USA 94:

  • 25.

    Henderson CE: Programmed cell death in the developing nervous system. Neuron 17:5795851997Henderson CE: Programmed cell death in the developing nervous system. Neuron 17:

  • 26.

    Hisahara SShoji SOkano Het al: ICE/CED-3 family executes oligodendrocyte apoptosis by tumor necrosis factor. J Neurochem 69:10201997J Neurochem 69:

  • 27.

    Hockenberry D: Defining apoptosis. Am J Pathol 146:16191995Hockenberry D: Defining apoptosis. Am J Pathol 146:

  • 28.

    Hockenberry DMOltvai ZNYin XMet al: Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:2412511993Cell 75:

  • 29.

    Horwitz HREllis HMSternberg DW: Programmed cell death in nematode development. Neurosci Comment 1:56651982Neurosci Comment 1:

  • 30.

    Jänicke RUSprengart MLWati MRet al: Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273:935793601997J Biol Chem 273:

  • 31.

    Kane AB: Redefining cell death. Am J Pathol 146:121995Kane AB: Redefining cell death. Am J Pathol 146:

  • 32.

    Katoh KIkata TKatoh Set al: Induction and its spread of apoptosis in rat spinal cord after mechanical trauma. Neurosci Lett 216:9121996Neurosci Lett 216:

  • 33.

    Keane RWSrinivasan AFoster LMet al: Activation of CPP32 during apoptosis of neurons and astrocytes. J Neurosci Res 48:1681801997J Neurosci Res 48:

  • 34.

    Kuida KZheng TSNa Set al: Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384:3683721996Nature 384:

  • 35.

    Li GIBrodin GFarooque Met al: Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord. J Neuropathol Exp Neurol 55:2802891996J Neuropathol Exp Neurol 55:

  • 36.

    Liu XZXu XMHu Ret al: Neuronal and glial apoptosis after traumatic spinal cord injury. J Neurosci 17:539554061997J Neurosci 17:

  • 37.

    Majno GJoris I: Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 146:3151995Am J Pathol 146:

  • 38.

    Martin SJGreen DR: Protease activation during apoptosis: death by a thousand cuts. Cell 82:3493521995Cell 82:

  • 39.

    Milligan CEPrevette DYaginuma Het al: Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro. Neuron 15:3853931995in vivo and in vitro. Neuron 15:

  • 40.

    Nagata S: Apoptosis by death factor. Cell 88:3553651997Nagata S: Apoptosis by death factor. Cell 88:

  • 41.

    Namura SZhu JFink Ket al: Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J Neurosci 18:365936681998J Neurosci 18:

  • 42.

    Nguyen KBMcCombe PAPender MP: Macrophage apoptosis in the central nervous system in experimental autoimmune encephalomyelitis. J Autoimmun 17:1451521994J Autoimmun 17:

  • 43.

    Nicotera PAnkarcrona MBonfoco Eet al: Neuronal necrosis and apoptosis: two distinct events induced by exposure to glutamate or oxidative stress. Adv Neurol 72:951011997Adv Neurol 72:

  • 44.

    Pender MPNguyen KBMcCombe PAet al: Apoptosis in the nervous system in experimental allergic encephalomyelitis. J Neurol Sci 104:81871991J Neurol Sci 104:

  • 45.

    Prineas JWKwon EEGoldenberg PZet al: Multiple sclerosis. Oligodendrocyte proliferation and differentiation in fresh lesions. Lab Invest 61:4895031989Lab Invest 61:

  • 46.

    Rink AFung KMTrojanowski JQet al: Evidence of apoptotic cell death after experimental traumatic brain injury in the rat. Am J Pathol 147:157515831995Am J Pathol 147:

  • 47.

    Savitz SIRosenbaum DM: Apoptosis in neurological disease. Neurosurgery 42:5555741998Neurosurgery 42:

  • 48.

    Shuman SLBresnahan JCBeattie NS: Apoptosis of microglia and oligodendrocytes after spinal cord injury in rats. J Neurosci Res 50:7988081997J Neurosci Res 50:

  • 49.

    Tator CHFehlings MG: Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 75:15261991J Neurosurg 75:

  • 50.

    Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science 267:145614621995Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science 267:

  • 51.

    Vela JMDalmau IGonzáles Bet al: The microglial reaction in spinal cords of jimpy mice is related to apoptotic oligodendrocytes. Brain Res 712:1341421996Brain Res 712:

  • 52.

    Weedon DSearle JKerr JF: Apoptosis. Its nature and implications for dermatopathology. Am J Dermatopathol 1:1331441979Am J Dermatopathol 1:

  • 53.

    Wood PL: Microglia as a unique cellular target in the treatment of stroke: potential neurotoxic mediators produced by activated microglia. Neurol Res 17:2422481995Wood PL: Microglia as a unique cellular target in the treatment of stroke: potential neurotoxic mediators produced by activated microglia. Neurol Res 17:

  • 54.

    Yakovlev AGKnoblach SMFan Let al: Activation of CPP32-like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury. J Neurosci 17:741574241997J Neurosci 17:

  • 55.

    Young W: Secondary injury mechanisms in acute spinal cord injury. J Emerg Med 11 (Suppl 1):13221993Young W: Secondary injury mechanisms in acute spinal cord injury. J Emerg Med 11 (Suppl 1):

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.

Headings

Figures

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

  • View in gallery

    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.

References

1.

Adams JC: Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40:145714631992Adams JC: Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40:

2.

Alnemri ESLivingston DJNicholson DWet al: Human ICE/CED-3 protease nomenclature. Cell 87:1711996 (Letter)Cell 87:

3.

Arends MJWyllie AH: Apoptosis: mechanisms and roles in pathology. Int Rev Exp Pathol 32:2232541991Int Rev Exp Pathol 32:

4.

Armstrong RCAja TJHoang KDet al: Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis. J Neurosci 17:5535621997J Neurosci 17:

5.

Barres BAJacobson MDSchmid R: Does oligodendrocyte survival depend on axons? Curr Biol 3:4894971993Curr Biol 3:

6.

Bauer JWekerle HLassmann H: Apoptosis in brain-specific autoimmune disease. Curr Opin Immunol 7:8398431995Curr Opin Immunol 7:

7.

Blight AR: Effect of 4-aminopyridine on axonal conduction-block in chronic spinal cord injury. Brain Res Bull 22:47521989Blight AR: Effect of 4-aminopyridine on axonal conduction-block in chronic spinal cord injury. Brain Res Bull 22:

8.

Bracken MBShepard MJCollins WFet al: A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the second National Acute Spinal Cord Injury Study. N Engl J Med 322:140514111990N Engl J Med 322:

9.

Bunge RPPuckett WRBecerra JLet al: Observations on the pathology of human spinal cord injury. A review and classification of 22 new cases with details from a case of chronic cord compression with extensive focal demyelination. Adv Neurol 59:75891993Adv Neurol 59:

10.

Cohen JJ: Apoptosis. Immunol Today 14:1261301993Cohen JJ: Apoptosis. Immunol Today 14:

11.

Crowe MJBresnahan JCShuman SL: Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nature Med 3:73761997Nature Med 3:

12.

Crowe MJShuman SLMasters JNet al: Morphological evidence suggesting apoptotic nuclei in spinal cord injury. Soc Neurosci Abstr 21:2321995 (Abstract)Soc Neurosci Abstr 21:

13.

Dusart ISchwab ME: Secondary cell death and the inflammatory reaction after dorsal hemisection of the rat spinal cord. Eur J Neurosci 6:7127241994Eur J Neurosci 6:

14.

Duvall EWyllie AHMorris RG: Macrophage recognition of cells undergoing programmed cell death (apoptosis). Immunology 56:3513581985Immunology 56:

15.

Ellis REYvan JHorvitz HR: Mechanisms and functions of cell death. Annu Rev Cell Biol 7:6636981991Annu Rev Cell Biol 7:

16.

Enari MTalanian RVWong WWet al: Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature 380:7237261996 (Letter)Nature 380:

17.

Fraser AEvan G: A license to kill. Cell 85:7817841996Cell 85:

18.

Furukawa KEstus SFu WMet al: Neuroprotective action of cycloheximide involves induction of Bcl-2 and antioxidant pathways. J Cell Biol 136:113711491997J Cell Biol 136:

19.

Gavrieli YSherman YBen-Sasson SA: Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:4935011992in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:

20.

Gehrmann JBanati RB: Microglial turnover in the injured CNS: activated microglia undergo delayed DNA fragmentation following peripheral nerve injury. J Neuropathol Exp Neurol 54:6806881995J Neuropathol Exp Neurol 54:

21.

Hall EDYonkers PAAndrus PKet al: Biochemistry and pharmacology of lipid antioxidants in acute brain and spinal cord injury. J Neurotrauma 9 (Suppl):S425S4421992J Neurotrauma 9 (Suppl):

22.

Hamburger V: Cell death in the development of the lateral motor column of the chick embryo. J Comp Neurol 160:5355461975Hamburger V: Cell death in the development of the lateral motor column of the chick embryo. J Comp Neurol 160:

23.

Hansebout RRBlight ARFawcett Set al: 4-Aminopyridine in chronic spinal cord injury: a controlled, double-blind, crossover study in eight patients. J Neurotrauma 10:1181993J Neurotrauma 10:

24.

Hara HFriedlander RMGagliardini Vet al: Inhibition of interleukin 1β converting enzyme family proteases reduce ischemic and excitotoxic neuronal damage. Proc Natl Acad Sci USA 94:200720121997Proc Natl Acad Sci USA 94:

25.

Henderson CE: Programmed cell death in the developing nervous system. Neuron 17:5795851997Henderson CE: Programmed cell death in the developing nervous system. Neuron 17:

26.

Hisahara SShoji SOkano Het al: ICE/CED-3 family executes oligodendrocyte apoptosis by tumor necrosis factor. J Neurochem 69:10201997J Neurochem 69:

27.

Hockenberry D: Defining apoptosis. Am J Pathol 146:16191995Hockenberry D: Defining apoptosis. Am J Pathol 146:

28.

Hockenberry DMOltvai ZNYin XMet al: Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:2412511993Cell 75:

29.

Horwitz HREllis HMSternberg DW: Programmed cell death in nematode development. Neurosci Comment 1:56651982Neurosci Comment 1:

30.

Jänicke RUSprengart MLWati MRet al: Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273:935793601997J Biol Chem 273:

31.

Kane AB: Redefining cell death. Am J Pathol 146:121995Kane AB: Redefining cell death. Am J Pathol 146:

32.

Katoh KIkata TKatoh Set al: Induction and its spread of apoptosis in rat spinal cord after mechanical trauma. Neurosci Lett 216:9121996Neurosci Lett 216:

33.

Keane RWSrinivasan AFoster LMet al: Activation of CPP32 during apoptosis of neurons and astrocytes. J Neurosci Res 48:1681801997J Neurosci Res 48:

34.

Kuida KZheng TSNa Set al: Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384:3683721996Nature 384:

35.

Li GIBrodin GFarooque Met al: Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord. J Neuropathol Exp Neurol 55:2802891996J Neuropathol Exp Neurol 55:

36.

Liu XZXu XMHu Ret al: Neuronal and glial apoptosis after traumatic spinal cord injury. J Neurosci 17:539554061997J Neurosci 17:

37.

Majno GJoris I: Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 146:3151995Am J Pathol 146:

38.

Martin SJGreen DR: Protease activation during apoptosis: death by a thousand cuts. Cell 82:3493521995Cell 82:

39.

Milligan CEPrevette DYaginuma Het al: Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro. Neuron 15:3853931995in vivo and in vitro. Neuron 15:

40.

Nagata S: Apoptosis by death factor. Cell 88:3553651997Nagata S: Apoptosis by death factor. Cell 88:

41.

Namura SZhu JFink Ket al: Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J Neurosci 18:365936681998J Neurosci 18:

42.

Nguyen KBMcCombe PAPender MP: Macrophage apoptosis in the central nervous system in experimental autoimmune encephalomyelitis. J Autoimmun 17:1451521994J Autoimmun 17:

43.

Nicotera PAnkarcrona MBonfoco Eet al: Neuronal necrosis and apoptosis: two distinct events induced by exposure to glutamate or oxidative stress. Adv Neurol 72:951011997Adv Neurol 72:

44.

Pender MPNguyen KBMcCombe PAet al: Apoptosis in the nervous system in experimental allergic encephalomyelitis. J Neurol Sci 104:81871991J Neurol Sci 104:

45.

Prineas JWKwon EEGoldenberg PZet al: Multiple sclerosis. Oligodendrocyte proliferation and differentiation in fresh lesions. Lab Invest 61:4895031989Lab Invest 61:

46.

Rink AFung KMTrojanowski JQet al: Evidence of apoptotic cell death after experimental traumatic brain injury in the rat. Am J Pathol 147:157515831995Am J Pathol 147:

47.

Savitz SIRosenbaum DM: Apoptosis in neurological disease. Neurosurgery 42:5555741998Neurosurgery 42:

48.

Shuman SLBresnahan JCBeattie NS: Apoptosis of microglia and oligodendrocytes after spinal cord injury in rats. J Neurosci Res 50:7988081997J Neurosci Res 50:

49.

Tator CHFehlings MG: Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 75:15261991J Neurosurg 75:

50.

Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science 267:145614621995Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science 267:

51.

Vela JMDalmau IGonzáles Bet al: The microglial reaction in spinal cords of jimpy mice is related to apoptotic oligodendrocytes. Brain Res 712:1341421996Brain Res 712:

52.

Weedon DSearle JKerr JF: Apoptosis. Its nature and implications for dermatopathology. Am J Dermatopathol 1:1331441979Am J Dermatopathol 1:

53.

Wood PL: Microglia as a unique cellular target in the treatment of stroke: potential neurotoxic mediators produced by activated microglia. Neurol Res 17:2422481995Wood PL: Microglia as a unique cellular target in the treatment of stroke: potential neurotoxic mediators produced by activated microglia. Neurol Res 17:

54.

Yakovlev AGKnoblach SMFan Let al: Activation of CPP32-like caspases contributes to neuronal apoptosis and neurological dysfunction after traumatic brain injury. J Neurosci 17:741574241997J Neurosci 17:

55.

Young W: Secondary injury mechanisms in acute spinal cord injury. J Emerg Med 11 (Suppl 1):13221993Young W: Secondary injury mechanisms in acute spinal cord injury. J Emerg Med 11 (Suppl 1):

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 4 4 4
Full Text Views 84 84 30
PDF Downloads 21 21 5
EPUB Downloads 0 0 0

PubMed

Google Scholar