(Stroke. 1997;28:2012-2017.)
© 1997 American Heart Association, Inc.
Articles |
From the Division of Cardiothoracic Surgery (M.E.M., G.K.K, N.T.K.) and Department of Neurology (Y.W., R.H., J.A.D., M.F.J., C.Y.H.), Washington University School of Medicine, St Louis, Mo.
Correspondence to Nicholas T. Kouchoukos, MD, 3009 N Ballas Rd, Room 266, St Louis, MO 63131.
| Abstract |
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Methods The infrarenal aorta of White New Zealand rabbits (n=24) was occluded for 40 minutes using a loop tourniquet. Rabbits were killed after 12, 24, or 48 hours (n=8 per group). The loop was placed but never tightened in sham-operated rabbits (n=6). The lumbar segment of the spinal cord (L5 to L7) was used for morphological studies, including hematoxylin and eosin staining and a modified terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining method. Electron microscopy was used to examine ultrastructural morphology. In addition, lumbar tissue was used for biochemical investigation of DNA laddering by agarose gel electrophoresis.
Results After ischemia, the affected areas
contained neurons with positive TUNEL staining. Positive neurons were
located in laminae III to IX, although most were concentrated in the
intermediate and ventral areas. Adjacent sections stained with
hematoxylin and eosin exhibited ischemic cell changes (red and
ghost neurons), while apoptotic bodies were also apparent. In
addition, electron microscopy of ischemic tissue samples
exhibited ultrastructural characteristics of apoptosis,
including nuclear condensation and relatively normal organelle
morphology. Finally, isolated DNA revealed a ladder on agarose gel
electrophoresis, indicating DNA fragmentation into
180 multiples of
base pairs.
Conclusions Spinal cord ischemia in rabbits induces morphological and biochemical changes suggestive of apoptosis. These data raise the possibility that apoptosis contributes to neuronal cell death after spinal cord ischemia.
Key Words: apoptosis neuronal death reperfusion spinal cord rabbits
| Introduction |
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Ischemic neuronal cell death has traditionally been attributed to necrosis.12 Recently, morphological studies have characterized two distinct types of cell death, namely, necrosis and apoptosis. Necrosis is characterized by compromised membrane integrity due to ATP energy depletion. A cell then swells, and the mitochondria become dysfunctional. Eventually the cell lyses, and lysosomal enzymes are released into the extracellular space. In comparison to changes in the cytoplasm, changes in the nucleus of a necrotic cell are relatively unremarkable, at least during the early stage. The nuclear membrane also swells as its materials scatter into small masses.13 In contrast to necrotic cells, apoptotic cells require energy and exhibit distinguishable changes in the nucleus, while initially maintaining cytoplasmic organelles.14 Subsequently, cytoplasmic structures degenerate, and both the nucleus and the cytoplasm may form membrane-bound "apoptotic bodies." Tissue macrophages eliminate the apoptotic bodies and dead cells, thereby causing little or no inflammatory response.15 16
Apoptosis is known to occur in the central nervous system during development and in pathological settings such ischemia.13 17 18 19 20 Apoptosis requires an active commitment of the cell to degrade its own DNA, according to an internal program of self-destruction.21 22 New protein synthesis is required for apoptosis, and protein synthesis inhibitors have been shown to reduce cell death postischemically.3 4 8 11 23 24 In contrast, necrosis is not a gene-facilitated process but results from injurious changes in the environment.
Nucleosomal fragmentation, which results in DNA fragments that are
multiples of
180 bp, is a well-defined biochemical marker of cells
undergoing apoptosis.13 15 25 These fragments are
produced by Ca2+-activated
endonucleases.13 Specific endonucleases have been linked
to nucleosomal DNA cleavage.16 26 In contrast, necrosis
causes nonspecific degradation of DNA into randomly sized
fragments.26 27 There is evidence for activation of the
apoptotic process in postischemic brain tissue; DNA
fragmentation has been described after both global and focal
ischemia in adult animals.3 4 8 9 10 11 28 29 30 31
Delayed neuronal degeneration appears to be more predominantly related
to apoptosis in a focal ischemic
model.11
Whether apoptosis contributes to delayed neuronal death after spinal cord ischemia/reperfusion remains unknown. In this study we investigated morphological and biochemical features of cell death in a rabbit model of spinal cord ischemia/reperfusion.
| Materials and Methods |
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Neurological status was scored by assessment of hindlimb neurological functions according to the modified Tarlov criteria.33 Behavioral analysis was conducted once a day. A score of 0 to 4 was assigned to each animal, as follows: 0, no voluntary hindlimb function; 1, movement of joints perceptible; 2, active movement but unable to stand; 3, able to stand but unable to walk; and 4, complete recovery of hindlimb function.
Light Microscopy
The rabbits were killed with the use of pentobarbital (120
mg/kg) followed by intracardiac perfusion of 1 L cold (0.9%)
NaCl 12, 24, and 48 hours (n=7 per group) after 40-minute
ischemia. In addition, sham rabbits (n=5) underwent the same
procedure. The lumbar segments of the spinal cord (L5 to L7) were
immediately extracted and flash fixed in 10% buffered formalin. They
were embedded in paraffin and cut (6 µm) for hematoxylin and
eosin staining and specific staining of DNA fragmentation
(deoxynucleotidyltransferase-mediated
dUTP-biotin nick end-labeling [TUNEL]) with the use of a molecular
biological-histochemical system (Apop Tag kit, Oncor). For the TUNEL
staining we used a model described by Du et al.11
Paraffin-embedded sections were deparaffinized in two changes of xylene
for 5 minutes each, then washed in 100%, 95%, and 70% ethanol,
respectively.11 Nuclei of tissue sections were stripped of
proteins by incubation with 20 µg/mL proteinase K (Sigma) for
5 minutes. Samples were washed in distilled water. We adapted a
modified TdT-mediated dUTP-biotin nick for 1 hour. The reaction was
ceased by incubating with the stop/wash buffer at 37°C for 30
minutes. After they were washed in phosphate-buffered saline, the
samples were incubated with the antidigoxigenin peroxide solution for
30 minutes. The samples were colorized with
DAB/H2O2 solution (0.2 mg/mL
diaminobenzidine tetrachloride and 0.005% H2O2
in 50 mmol/L Tris-HCl buffer) and then counterstained with
methyl green. For controls, samples were treated similarly, but
distilled water was substituted for TdT enzyme.
Electron Microscopy
An animal from each group (n=4) was killed and transcardially
perfused with 200 mL of saline containing 1% heparin, followed by 2%
glutaraldehyde and 2% paraformaldeyde in phosphate
buffer (pH 7.6). After perfusion for approximately 20 minutes, L5 to L7
of the spinal cord was removed, and slabs (
500 m) were placed in the
perufasate overnight at 4°C. Slabs were washed the next day in
phosphate buffer and then osmicated with 1% osmium tetroxide in
phosphate buffer for 1 hour. Slabs were dehydrated in a graded ethanol
series. Infiltration was accomplished with the use of a series of
propylene oxide and EmBed 812 (EM Sciences) mixtures. Slabs were left
in 100% EmBed 812 for 6 hours. Finally, slabs were flat embedded
between ACLAR sheets (Ted Pella) at 60°C overnight.
Slabs were glued to blank blocks, and ultrathin sections (60 to 90 nm) were cut on a Reichart Jung Ultracut E with a diamond knife. Ultrathin sections were collected on copper grids and stained with 3% uranyl acetate in 50% ethanol for 20 minutes followed by lead citrate for 3 minutes. Sections were visualized and photographed on a JEOL 100CX electron microscope.
Gel Electrophoresis
Adjacent sections of tissue (L5 to L7) from all rabbits used for
light microscopy were flash frozen in liquid nitrogen and stored at
-75°C. Spinal cord DNA was prepared according to the methods
described by Sambrook et al.34 Briefly, 100 mg of fresh
spinal cord was homogenized in an Eppendorf tube containing
600 µL cell lysis TE buffer (10 mmol/L Tris-HCl, pH 8.0,
100 mmol/L EDTA, 20 µg/mL 0.5% sodium
dodecyl sulfate) with the use of a Kontes Pellet Pestle
(Fisher). After it was mixed with additional 600 µL cell lysis
buffer, the homogenized sample was incubated at 65°C for
1 hour and incubated at 55°C with proteinase K (final concentration
100 µg/mL, Sigma) overnight. Sample was extracted with an
equal volume of phenol/chloroform/amyl (25:24:1). Total DNA
contained in aqueous phase was precipitated with ethanol. DNA pellet
was washed with 70% ethanol twice and dissolved in 50 µL TE buffer.
The DNA was then treated with DNAase-free RNAase (10 u/mL, Sigma) at
37°C for 1 hour. DNA concentration was determined by measurement of
OD260. Equal amounts of DNA samples were used in 1.5% agarose
electrophoresis with TAE buffer (40 mmol/L Tris-acetate,
1 mmol/L EDTA, pH 8.0). DNA was then visualized with
ethidium bromide.
| Results |
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Hematoxylin and eosin staining of the spinal cord of sham-operated
rabbits showed no signs of neuronal damage. Necrotic changes,
eosinophilic cytoplasm (red neuron) and ghost neurons, and neuropil
vacuolation of the gray matter (laminae III to IX) were evident after
24 and 48 hours (Fig 1A
) Among these
cells, apoptotic-like bodies were seen but were more
conspicuous in TUNEL staining, as described below. Infiltration by
monocytes and neutrophils was noted at 24 and 48 hours.
|
Samples from the sham-operated animals showed no evidence of TUNEL
staining (Fig 1B
); however, when treated with DNAase sham samples, they
produced many positively stained nuclei (data not shown). Replacement
of distilled water for TdT enzyme yielded negative controls. A few
scattered cells in the gray matter were positive for TUNEL staining 12
hours after ischemia. The number of TUNEL-positive cells
reached a peak at 24 hours (Fig 1C
) and decreased at 48 hours. The
apoptotic cells were scattered within areas that contained
necrotic cells. They were most highly concentrated in laminae III
through IX and were observed more in the intermediate to ventral areas
of the gray matter. Many cells positive for TUNEL staining revealed
nuclear or cytoplasmic budding, known as apoptotic bodies (Fig 1D
).
Sham samples revealed normal cell structure on electron microscopy (Fig 2A
). Necrotic cells exhibited nuclear and
cytoplasmic disintegration with breakdown of organelle membranes (Fig 2B
). Cells exhibiting apoptotic ultrastructural features were
identified at all three time points, revealing typical
apoptotic morphology (the cell is shrunken, the chromatin has
condensed around the margin of the nucleus, and the cytoplasm contains
large vesicles, while the mitochondria appear normal) (Fig 2C
).35 36 Note the necrotic cell alongside the
apoptotic cell. In the late stage of apoptosis, nuclear
material becomes extremely condensed in small masses (Fig 2D
) and will
bud into apoptotic bodies.
|
At 24 hours after ischemia, a ladder of multiples of
180-bpsized fragments appeared (Fig 3
). At 48 hours, a faint ladder also
appeared but was not reproduced well on film.
|
| Discussion |
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The effects of ischemia and reperfusion on the progression of neural death are complex. While early reperfusion can limit the extent of necrosis, reperfusion may also exert a variety of potentially deleterious effects that are collectively described as reperfusion injury. It is possible that during reperfusion certain neurons may recover energy metabolism but then go on to die between hours and several days later, indicating delayed cell death.37 Apoptosis has been shown to be a feature of reperfusion injury in cardiac myocytes, leading to late cell death.38 39 However, the precise sequence of intracellular events during reperfusion is poorly understood. Mild and severe ischemia/reperfusion stimuli have been shown to initiate apoptosis.9 Furthermore, ischemia has been shown to initiate an active ongoing process of apoptotic cell death.40
Apoptotic cells also accompany necrotic cells, as seen on electron microscopy and light microscopy, raising the possibility that apoptosis is initiated by the same stimuli that cause necrosis. It has been shown that 2 hours of middle cerebral artery ischemia elicits not only necrosis but also DNA fragmentation indicative of apoptosis.40 DNA electrophoresis of the ischemic/reperfused spinal cord samples revealed a ladder as well as a smear, indicating that both necrosis and apoptosis are occurring at the same time. Previous findings have shown DNA fragmentation and smearing from the ischemic hemisphere after focal ischemia and from the hippocampal CA1 region after forebrain ischemia.8 40 41 It is possible that some cells may begin to die by apoptosis but then acquiesce to necrosis as the environment becomes more toxic.9 Alternatively, apoptosis may represent a secondary injury process as a consequence of necrosis. It is possible that some cells exposed to ischemia can detect the injury and trigger a signaling process to die by apoptosis rather than necrosis. Apoptosis may avoid the inflammatory reaction that may develop after necrotic cell death.
Duval and Wyllie42 showed that mild membrane damage, such as that caused from ischemia, might allow influx of Ca2+ sufficient to initiate apoptosis but insufficient to extensively activate phosolipases leading to necrosis. What causes the cell to be "at risk" for either necrosis or apoptosis has yet to be understood. However, all cells of the spinal cord are not affected at the same time despite being subjected to the same treatment. Therefore, irregular distribution of apoptosis may be due in part to differences in loss of perfusion and reflow due to microcirculation plugging, metabolism, and differential vulnerability to ischemic insult.
Apoptotic cells are identifiable on hematoxylin and eosin staining but are more apparent with TUNEL staining. Although some necrotic neurons contained stainable amounts of DNA, apoptotic cells are more intensely stained and often exhibited apoptotic bodies. Electron microscopy reveals compaction of chromatin against nuclear membrane, nuclear segregation, cell shrinkage with preservation of organelles, detachment from surrounding cells, and nuclear condensation. Mitochondria, endoplasmic reticulum, and the Golgi apparatus appear relatively normal in the early stages of apoptosis. Cytoplasmic shrinkage and eventual budding off to form membrane-bound fragments, known as apoptotic bodies, follow.15 43
Since morphological and ultrastructural appearances are open to varying interpretations, the detection of a DNA ladder on gel electrophoresis adds biochemical evidence of DNA fragmentation. The morphological evidence and the visualization of a DNA ladder indicate that apoptosis is occurring postischemically. It is apparent in the gels that specific degradation is occurring concurrently with nonspecific degradation. Light microscopy and electron microscopy affirm this. Together, these data suggest that apoptosis occurs concurrently with necrosis.
Although apoptotic morphology has been associated with certain biochemical features, it is not possible to conclude on the basis of morphology that spinal cord ischemia activates a process identical to that which occurs during development or to infer the nature of the biochemical process underlying apoptosis.13 25 In addition, the role played by the intensity of ischemia in determining the induction of apoptosis is not understood. We cannot conclude that apoptosis is the sole method of death, but it plays an important role in cell death after spinal cord ischemia.
Cycloheximide has been shown to reduce delayed neuronal degeneration after focal brain ischemia.8 11 Preliminary studies using a traumatic spinal cord injury model also showed a neuroprotective effect of cycloheximide.44 These results suggest that therapeutic intervention such as cycloheximide, a protein synthesis inhibitor, might be valuable in reducing cell death after spinal cord ischemia. One interesting implication of these findings is that apoptosis may help to explain clinical cases of delayed-onset paraplegia and furthermore may suggest that apoptosis is a potentially preventable process.45
| Acknowledgments |
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Received April 9, 1997; revision received June 5, 1997; accepted June 18, 1997.
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L. Lang-Lazdunski, C. Heurteaux, N. Vaillant, C. Widmann, and M. Lazdunski RILUZOLE PREVENTS ISCHEMIC SPINAL CORD INJURY CAUSED BY AORTIC CROSSCLAMPING J. Thorac. Cardiovasc. Surg., May 1, 1999; 117(5): 881 - 889. [Abstract] [Full Text] [PDF] |
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