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(Stroke. 1997;28:2012-2017.)
© 1997 American Heart Association, Inc.

Articles

Cell Death Suggestive of Apoptosis After Spinal Cord Ischemia in Rabbits

Mary E. Mackey, BA, PA-S; Yingji Wu, MSc; Rong Hu, MD; Joseph A. DeMaro, MA; Mark F. Jacquin, PhD; Georgios K. Kanellopoulos, MD; Chung Y. Hsu, MD, PhD; Nicholas T. Kouchoukos, MD

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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Background and Purpose After spinal cord ischemia, some neurons remain viable after an ischemic insult but may be at risk of dying during reperfusion. We searched for morphological and biochemical features of apoptosis, which is a mechanism of delayed neuronal death, in a rabbit model of spinal cord ischemia.

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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One of the most serious complications of surgery for thoracoabdominal aneurysms is paraplegia, with an incidence rate ranging from 4% to 33%.¹ It is believed that the cause of spinal cord dysfunction is ischemia from hypoperfusion during cross-clamping. However, some patients awake with no neurological dysfunction only to develop delayed-onset paraplegia 1 to 5 days later. The cause of delayed-onset paraplegia is poorly understood, but it has been attributed to postoperative hypotension, embolization or thrombosis to anterior spinal artery, and occlusion of reimplanted intercostal artery.² Research has also indicated other factors as mediators of delayed cell death after central nervous system ischemia, including free radical production, deleterious effects of leukocytes and microglia, and apoptosis.^{3 4 5 6 7 8 9 10 11}

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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White New Zealand rabbits weighing 3 to 4 kg were employed to produce a reliable model of paraplegia after spinal cord ischemia with the use of the model described by Zivin and DeGirolami.³² The animals were fasted for 12 hours. Sedation was induced by ketamine (20 mg/kg), and the animals were intubated and anesthetized with 1.5% to 2.5% isoflurane. Ventilation was set to maintain arterial PCO₂ between 33 and 38 mm Hg. Arterial blood pressure was monitored through cannulas placed in the femoral and carotid arteries and was maintained at 100 mm Hg. Rectal temperature was monitored and was maintained at 39±1°C with heating pads. A laparotomy was performed, the infrarenal aorta was isolated, and a loop tourniquet was placed around it. Ischemia was induced in 24 rabbits by tightening of the tourniquet for 40 minutes. Six sham rabbits underwent the same operative procedure, but the tourniquet was not tightened. The animals were extubated 10 to 15 minutes after the release of the tourniquet and were killed at 12, 24, or 48 hours (n=8 per ischemic group). Sham animals (n=6) were killed at 24 hours. Animals that developed urinary retention had decompression of the bladder performed according to the Credé maneuver.

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 anti–digoxigenin 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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All experimental animals were completely paraplegic (score of 0) postoperatively, and no improvement was seen during the observation periods, while all sham animals maintained normal motor behavior (score of 4). All rabbits survived without major complications.

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.

View larger version (94K): [in this window] [in a new window]   Figure 1. Horizontal paraffin sections (6 µm) of lumbar cord stained with hematoxylin and eosin (A) and stained with Apop Tag kit and counterstained with methyl green (B through D). A, Typical necrotic changes include eosinophilic cytoplasm or red neuron (arrow). Apoptotic bodies (open arrows) are also apparent (original magnification x440). B, Sham sample reveals no deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining of neurons (arrow) 24 hours after 0 minutes of spinal cord ischemia (original magnification x440). C, These TUNEL-positive neurons (arrow) reveal DNA fragmentation 24 hours after 40 minutes of spinal cord ischemia. The TUNEL staining method tags the multitude of 3' ends after endonuclease activity (original magnification x440). D, Apoptotic bodies (arrow) are apparent 24 hours after 40 minutes of spinal cord ischemia with TUNEL staining (original magnification x550).

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.

View larger version (150K): [in this window] [in a new window]   Figure 2. Electron microscopy of lumbar spinal cord control. A, Nuclear chromatin is evenly distributed (arrow) 24 hours after 0 minutes of spinal cord ischemia in the control (sham) (original magnification x10 000). B, A necrotic neuron (arrow) revealing cytoplasmic and nuclear disintegration with breakdown of organelles 24 hours after 40 minutes of spinal cord ischemia (original magnification x10 000). C, Chromatin has condensed around the nuclear margin (arrow); cytoplasmic organelles remain relatively normal in morphology 24 hours after 40 minutes of spinal cord ischemia. Note necrotic cell (open arrow) alongside (original magnification x10 000). D, In late-stage apoptosis, neuron reveals irregularly shaped nuclei with dense chromatin masses (arrow) 48 hours after 40 minutes of spinal cord ischemia (original magnification x10 000).

At 24 hours after ischemia, a ladder of multiples of 180-bp–sized fragments appeared (Fig 3). At 48 hours, a faint ladder also appeared but was not reproduced well on film.

View larger version (71K): [in this window] [in a new window]   Figure 3. Internucleosomal DNA laddering detected by gel electrophoresis 24 hours after 40 minutes of spinal cord ischemia. Photograph of ethidium bromide–stained gel. Lane 1 is a DNA standardized 1-kb ladder. Lane 2 is a control from a sham animal. Lane 3 exhibits DNA fragmentation into 180 bp after 40 minutes of spinal cord ischemia and 24 hours of reperfusion.

	Discussion

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After spinal cord ischemia, certain cells may recover metabolic activity upon reperfusion but may die 24 to 48 hours later. Our results suggest that this delayed neural death associated with spinal cord ischemia may be due in part to apoptosis. Forty minutes of spinal cord ischemia in the rabbit evokes DNA fragmentation and chromatin condensation, indicative of apoptosis. We estimate that approximately 30% to 40% of the cells show features of apoptosis by 24 hours after ischemia.

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

 
This study was supported in part by The Shoenberg Foundation; the Lifeline Foundation; National Institutes of Health grants 1 T32 HL-07776, DE-07734, NS-25545, and NS-28995; and Office of Naval Research grant N-00014-95-1-0582. The authors would like to express their thanks to Du Cheng for helpful discussion and Donna Hand for technical assistance.

Received April 9, 1997; revision received June 5, 1997; accepted June 18, 1997.

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