- Email: [email protected]
Temporal–Spatial Pattern of Acute Neuronal and Glial Loss after Spinal Cord Contusion
Experimental Neurology 168, 273–282 (2001) doi:10.1006/exnr.2001.7628, available online at http://www.idealibrary.com on
Temporal–Spatial Pattern of Acute Neuronal and Glial Loss after Spinal Cord Contusion S. D. Grossman, L. J. Rosenberg, and J. R. Wrathall Department of Cell Biology and Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road, Washington, DC 20007 Received April 11, 2000; accepted December 19, 2000
The secondary loss of neurons and glia over the first 24 h after spinal cord injury (SCI) contributes to the permanent functional deficits that are the unfortunate consequence of SCI. The progression of this acute secondary cell death in specific neuronal and glial populations has not previously been investigated in a quantitative manner. We used a well-characterized model of SCI to analyze the loss of ventral motoneurons (VMN) and ventral funicular astrocytes and oligodendrocytes at 15 min and 4, 8, and 24 h after an incomplete midthoracic contusion injury in the rat. We found that both the length of lesion and the length of spinal cord devoid of VMN increased in a time-dependent manner. The extent of VMN loss at specified distances rostral and caudal to the injury epicenter progressed symmetrically with time. Neuronal loss was accompanied by a loss of glial cells in ventral white matter that was significant at the epicenter by 4 h after injury. Oligodendrocyte loss followed the same temporal pattern as that of VMN while astrocyte loss was delayed. This information on the temporal–spatial pattern of cell loss can be used to investigate mechanisms involved in secondary injury of neurons and glia after SCI. © 2001 Academic Press Key Words: cell death; motoneurons; glia; electron microscopy; apoptosis; necrosis; GFAP; CC1; morphometry.
INTRODUCTION
In recent studies on experimental spinal cord injury (SCI), we observed that loss of large ventral horn motor neurons (VMN) adjacent to the thoracic injury site is extensive by 24 h after SCI with no evidence of significant additional cell loss of VMN 1 month later (15). The first 24 h after injury appears critical for loss of VMN. The loss of these neurons is functionally significant since their preservation through acute treatment with a neurotrophic factor results in improved respiratory function (36). We have also examined the development of white matter pathology and axonal loss that
occurs in ventromedial white matter in the first 24 h after SCI (32), which includes loss of glial cells. The oligodendrocyte loss at 24 h after injury can be significantly reduced by acute treatment with the AMPA/ kainate antagonist, NBQX (30), a treatment that results in increased chronic preservation of white matter and reduced long-term hind limb functional deficits (40). Thus, the loss of white matter glia in the first 24 h after SCI is also an important aspect of the secondary injury seen after spinal cord contusion. There have been a number of studies on histopathology after SCI that have used morphometric techniques to quantify tissue loss in gray and white matter (3, 6, 7, 25, 26). Recently a number of studies have focused on the occurrence of apoptosis in cells after SCI (1, 12, 21, 22, 38) and have provided quantitative information on the time course and location of cells demonstrating markers of apoptosis (21). However, to our knowledge there has been no previous quantitative study of the overall temporal–spatial pattern of loss of specific populations of cells after SCI. We therefore used a standardized and well-characterized rat model of incomplete SCI produced with a weight drop device (41) to assess the pattern of loss of VMN and ventromedial white matter glia at acute times after SCI. Loss of VMN, astrocytes, and oligodendrocytes at specific distances from the injury epicenter was quantified using a combination of immunohistochemistry and light and electron microscopy at 15 min, 4 h, 8 h, and 24 h after SCI. An accompanying study illustrates how these quantitative time-course data can be used to evaluate a potential mechanism of cell death involving alterations in glutamate receptor subunit expression (42). MATERIALS AND METHODS
Spinal cord injury. SCI was performed as previously described (15, 41). A total of 56 female, Sprague– Dawley rats weighing 200 –250 g were used for the studies. Animals were housed on a 12-h light/dark cycle with standard rat chow and water ad libitum.
273
0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
274
GROSSMAN, ROSENBERG, AND WRATHALL
Rats were anesthetized with chloral hydrate (360 mg/kg ip), and a laminectomy was performed at T8, exposing the underlying dura. The spinal column was stabilized with angled Allis clamps on the T7 and T9 spinous processes. The impounder tip of a weight drop device (2.4 mm in diameter) was lowered onto the dura and a 10-g weight was dropped from a height of 2.5 cm onto the impounder to produce an incomplete spinal contusion. Controls for these experiments were age-, sex-, and weight-matched laminectomized rats. This model has been characterized in terms of biomechanics, resulting functional deficits, somatosensory evoked potentials, and quantitative histopathology (14, 25–27, 29). Histopathology. Spinal cords were rapidly removed from control and SCI rats (n ⫽ 5 at each time point) and 1.5-mm segments of thoracic cord, centered on the injury site, were quick frozen on dry ice and embedded in OCT (Tissue-Tek, Torrance, CA). Spinal cord segments were embedded in sets consisting of one or two segments from injured animals and one segment from an uninjured control and were sectioned at 20 m on a Jung Frigocut 2800 E cryostat. Sections were thaw mounted onto Superfrost Plus slides (Fisher) such that each slide represented 100 m of tissue and stored at ⫺20°C until used. Slides representative of each millimeter length of cord were stained with eriochrome RC (28), counterstained with hematoxylin and eosin, and used to reconstruct the injury site, as previously described (37). Lesion length was determined by tracing stained sections from each millimeter of spinal cord using a projector. We counted the numbers of sections exhibiting disruption of tissue and/or presence of hemorrhage to get a final lesion length in millimeters. Slides corresponding to the lesion epicenter and specific distances rostral and caudal to the epicenter were used in determining cell loss. Criteria for determination and quantification of ventral motoneurons. VMN were identified by their presence in the lower ventral horn, below the deep apex where the ventromedial gray and white matter meet, about 140 m below the central canal. Cells above this line were excluded. Counts were also based on size criteria. Specifically, VMN were counted by central profile counts (11) with use of a reticule that was divided into squares measuring 0.02 ⫻ 0.02 mm. Only VMN with diameter larger than half of one square were counted, with those counted having diameters ranging from 30 to 70 m. Inclusion also required the presence of a prominent nucleolus to avoid counting the same cell twice. Three slides were selected for each millimeter distance from the epicenter, each slide having 5 sections of spinal cord, for a total of 15 sections from each animal. The numbers of VMN per horn were counted in 10 sections, selected at random, according to
central profile counting procedures (11), and the average per horn was calculated. Immunohistochemical identification and quantification of glia. Slides adjacent to those used for VMN counts were labeled with antibody to glial fibrillary acid protein (GFAP; DAKO Corp., Carpinteria, CA) to identify astrocytes or CC1 (APC-7; Oncogene Research Products, Cambridge, MA) antibody, which recognizes the adenomatous polyposis coli gene expressed in oligodendrocytes (4, 12, 34). Primary antibodies were used at a dilution of 1:100 for GFAP and 1:1000 for CC1. CC1 used at this concentration is selective for identifying oligodendrocytes as determined by double immunolabeling with astrocyte and other oligodendrocyte antigens (4, 34). Endogenous peroxidase was quenched with 0.3% hydrogen peroxide in 0.1 M Tris buffer (pH 7.4) for 20 min. Sections were blocked for 1 h with 5% bovine serum albumin in 0.1 M Tris buffer. The blocking serum was removed and the tissue was exposed overnight at 4°C to the primary antibodies. The next day, the tissue was washed with Tris buffer and exposed to the secondary antibody for 30 – 45 min. Labeling was visualized using the ABC peroxidase technique (Vector Laboratories, Inc., Burlingame, CA) with 3,3⬘-diaminobenzidine or the VIP substrate kit (Vector Laboratories, Inc.). Sections were counterstained with hematoxylin or methyl green (Vector) for the purpose of determining total nuclei present. Counts of labeled glial cells were performed in a 0.2-mm 2 area of ventromedial white matter. Two slides representative of the lesion epicenter and specific millimeter distances rostral and caudal to it were examined. The first and fifth section from the five sections on each slide were used for quantification purposes. Only intensely labeled cells with a clearly defined nucleus were used to establish numbers of astrocytes and oligodendrocytes. Hematoxylin counts were performed at the same time to determine numbers of total nuclei. Electron microscopy. At 4 and 24 h after SCI, rats (n ⫽ 5 per group) were anesthetized with 4% chloral hydrate and intracardially perfused with saline followed by 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4. The spinal cords were removed from the vertebral column and fixed overnight in fresh fixative. A 2-cm segment, centered on the epicenter of the injury, was cut from each cord and embedded in 4% agar. The embedded tissue blocks were mounted on a tissue chopper stage (Sorvall, Newtown, CT) and cut into 250-m cross sections (4 sections ⫽ 1 mm of tissue). Tissue sections from the epicenter and from 1 mm rostral and caudal to the epicenter were used in studies of acute axonal pathology (30, 31). For the current study, sections at 2 and 4 mm rostral and caudal to the epicenter were postfixed for 1 h in 1% osmium–1% potassium ferricyanide, en bloc stained with 1% uranyl acetate and flat-embedded in
NEURONAL AND GLIAL DEATH AFTER SCI
Spurr resin (Ted Pella, Inc., Redding, CA). One-micrometer sections were cut from each tissue block, stained with 1% toluidine blue, and viewed with light microscopy. Blocks were trimmed to approximately 1 mm 2 in areas of interest and ultrathin (70 –90 nm) sections were cut and placed on 200-mesh nickel grids (Ted Pella, Inc.). Sections were viewed with a JEOL Jem 1200 EX (Tokyo, Japan) transmission electron microscope. Statistical analysis. The averages for each rat at each location were used to calculate means and standard errors for the group (n ⫽ 5 rats) at each time point examined. Statistical significance was determined using SigmaStat, a computerized statistics program (SPSS, San Raphael, CA). A one- or two-factor analysis of variance (ANOVA) followed by a post hoc test (Tukey) was performed with P values set at 0.05 to establish statistical differences between specific groups or time points. RESULTS
We examined spinal cord tissue from animals sacrificed 15 min or 4, 8, or 24 h after SCI or laminectomy. In spinal cords from laminectomy controls, we saw tightly packed cells in the gray matter with healthy appearing VMN (Fig. 1A). Gray matter was clearly delineated from white matter that contained structurally intact glia and axons. At 15 min after injury at the epicenter, many VMN were lost and the tight network of cells seen in controls was abolished (Fig. 1B). Distinction between gray and white matter was still apparent. At 4 and 8 h after SCI, there was virtual destruction of the ventral horns at the epicenters. At 8 h, white matter and gray matter were no longer clearly distinguishable due to tissue disruption and degeneration (Fig. 1C). At 24 h after injury, this disruption became even more apparent, and large areas of tissue were completely lost (Fig. 1D). Close examination of ventral horns showed there was complete loss of VMN by 24 h at the epicenter. This progressive destruction of the cord following SCI was seen not only at the lesion epicenter but also rostral and caudal to it. At 15 min tissue disruption appeared localized mainly in the area of direct mechanical impact (epicenter ⫾ 1 mm) with hemorrhage, primarily in dorsal funicular white matter, extending farther rostral and caudal to the impact site. At 2 mm from the epicenter, viable VMN and intact ventral white matter were still present. Tissue disruption was evident only in central gray and dorsal white matter, leaving the majority of the cord morphologically intact. The lesion expanded radially and longitudinally by 4 and 8 h, with further destruction of gray matter and mild to moderate disruption of white matter adjacent to the gray matter. By 24 h (Fig. 1E) a large cavity was formed at the epicenter, surrounded by a rim of intact
275
peripheral white matter. At distances of ⫾2 mm from epicenter, gray matter was still present, though clearly altered by the injury. Viable VMN were rarely seen. While visibly disrupted, a considerable amount of white matter remained. At 4 mm rostral and caudal, tissue damage was localized to the dorsal funicular white matter (Fig. 1E). At this location the gray matter appeared remarkably intact 24 h after SCI with a number of viable VMN seen, and white matter displayed little, if any, damage by light microscopy. Examination of residual ventral white matter at 24 h at the epicenter showed numerous hematoxylinstained glial bodies with abnormal nuclei characterized by nuclear condensation and fragmentation (Fig. 1G). Normal glial nuclei in laminectomy controls appeared as small, round structures with a smooth, intact edge (Fig. 1F). In order to visualize entire cells and to distinguish between subtypes of glia, immunolabeling was performed. Normal oligodendrocytes stained with CC1 displayed large cell bodies, dark and evenly stained cytoplasm (Fig. 1H). Lightly stained processes could be seen radiating short distances from the cell body. Oligodendrocytes at 24 h after SCI lacked radiating processes and displayed punctate dark aggregates in the cytoplasm and nucleus (Fig. 1I). Astrocytes labeled with GFAP also demonstrated striking morphological changes at 24 h after SCI (Fig. 1K). Normal astrocytes had small, intensely labeled cell bodies and numerous long processes extending from the perikaryon cell (Fig. 1J). After injury, these processes appeared discontinuous and the perikarya had rounded (Fig. 1K). Labeling intensity in the cell bodies was greatly diminished but astrocyte processes displayed increased immunoreactivity To better evaluate morphological changes occurring in VMN and glia after injury, 1-m plastic sections of spinal cord tissue were examined 4 and 24 h after SCI at distances of 2 and 4 mm from the epicenter. Neuronal pathology observed was consistently indicative of necrotic cell death, with no evidence of apoptosis seen at the time points examined. Neurons showed extensive and variable pathology (Figs. 2A–2D). Many of the neurons looked pyknotic, with vacuolization either in the nucleus or in the cytoplasm (Figs. 2A, 2B, and 2D). Others neurons appeared swollen and faintly stained, resulting in a “ghostly” appearance (Fig. 2C). The nuclei of the moribund VMN ranged from a large, swollen appearance with intact nuclear envelope (Fig. 2C) to a dark, condensed structure. Further, nucleoli also exhibited a variety of pathologies. While some nucleoli looked small and condensed (Fig. 2C), others appeared swollen and large (Fig. 2B), while others were fragmented (Fig. 2D). Although most of the neuronal pathology we observed was more consistent with a necrotic, rather than an apoptotic, phenotype, this was not the case in glia (Fig. 3). In general, most glia in the ventromedial
276
GROSSMAN, ROSENBERG, AND WRATHALL
FIG. 1. Microscopic examination of tissue pathology after SCI. Bright-field micrographs of spinal cord ventral horn at 20⫻ magnification showing a normal ventral horn (A) and ventral horns at 15 min (B), 8 h (C), and 24 h after SCI (D). Arrows show VMN included for cell counts, fitting size criteria and displaying a nucleolus. Arrowhead points to VMN excluded from analysis. Scale bar, 0.175 mm. (E) Photographs displaying a typical spinal cord lesion at 24 h after injury. The center section represents lesion epicenter. Sections left and right of the center section represent spinal cord sections at 2 mm rostral and caudal to the epicenter. Sections to the far right and left are taken at distances of 4 mm rostral and caudal to the epicenter. Scale bar, 0.5 mm. (F–K) Glial pathology at 24 h after SCI at the epicenter compared to normal controls. Ventromedial white matter stained with hematoxylin was used to visualize normal (F) and pathological (G) nuclei at 24 h after SCI. Oligodendrocytes or astrocytes were identified with the antibodies CC1 and GFAP. Photographs represent typical pathological changes seen in oligodendrocytes (I) and astrocytes (K) at 24 h after SCI at the lesion epicenter, compared to their respective controls (H and J).
white matter after SCI appeared swollen. In many, the nuclear chromatin appeared in dispersed clumps throughout the nucleus. In other cells, the nuclei were
irregularly shaped, with a single condensed aggregate of chromatin. Glial apoptosis was confirmed by electron microscopy at 24 h after SCI (Fig. 3C).
NEURONAL AND GLIAL DEATH AFTER SCI
277
FIG. 2. Selected ventral motor neurons show various pathologies after SCI. Moribund VMN were photographed at 100⫻ magnification at 2 mm from the epicenter at 4 h after SCI (A, C, D) or at 4 mm from the epicenter at 24 h after SCI (B). Extensive and variable pathology can be seen in 1-m plastic sections of ventral gray matter stained with toluidine blue.
Quantification of neurons. Having observed a progressive spread of injury rostral and caudal to the impact site, slides representing sequential millimeter distances distal to the epicenter were selected for quantitative lesion analysis at 15 min or 4, 8, or 24 h after SCI (Fig. 4). At 15 min the average length of the lesions (defined as presence of tissue disruption and/or hemorrhage) was about 9 mm. There was a progressive increase in lesion length over time. By 24 h the lesion length averaged about 13 mm, representing a 30% increase from 15 min postinjury (Fig. 4). There was also a progressive loss of residual VMN. Control rats had an average of approximately 10 VMN per horn (based on criteria described under Materials and Methods) at all thoracic locations examined (Fig. 5). At 15 min after SCI, only 33% of the normal number of VMN were present at the epicenter of injury. At ⫾1 mm from the epicenter 48% of control VMN were still present. At ⫾2 and 3 mm, however, approximately 50 and 60% of VMN remained, respectively. By ⫾4 mm, however, the numbers of VMN in injured rats was not statistically different from control numbers, averaging 91% of control values. VMN counts in rats sacrificed 4 and 8 h after SCI (Fig. 5) showed no difference between the two experimental groups, although they were significantly different from controls at all distances examined (Fig. 5). Complete VMN loss was seen at epicenter. At 4 mm
FIG. 3. Glial pathology as seen by microscopic examination of ventromedial white matter. (A) Swollen glia were typically found at 4 h after SCI. Nuclei demonstrated a grainy appearance (arrowhead) or contained clumped chromatin (arrow). Generally, the nuclear envelope was intact. (B) Many glia had irregular nuclei and were dark in appearance (arrowheads). Typically, the nuclear envelope of these cells was rough or ragged in appearance as opposed to the smooth nuclear envelope seen in glia with swollen nuclei (A). (C) High-magnification (2000⫻) showed that a number of glia had an apoptotic phenotype at 24 h after SCI as demonstrated by the presence of compartmentalized, chromatin bodies within the cytoplasm.
278
GROSSMAN, ROSENBERG, AND WRATHALL
FIG. 4. Lesion length increases with time after SCI. Graph represents quantification of the length of spinal cord lesion in rats sacrificed 15 min or 4, 8, or 24 h after SCI. The length of lesion increases over time after the injury, to reach significantly longer lengths at 24 h compared to lesion length observed at 15 min postinjury. N ⫽ 5 rats per group. Bars represent means ⫾ SE. *Significant difference compared to 15-min time point. Statistical significance was determined with a Tukey post hoc test with P ⬍ 0.05.
distal, injured rats had 77% of control numbers of VMN. By 4 and 8 h, there was approximately a 2.5- to 3-mm segment of cord lacking VMN. By 24 h, tissue destruction was so severe that VMN were completely lost at the injury epicenter and 2 mm in either direction. The survival rate at 4 mm rostral and caudal was approximately 44% and the length of cord devoid of VMN measured 4 mm (Fig. 5). At each time point we examined, the loss of VMN was symmetrical rostral and caudal to the injury epicenter. It is important to note that in this model there is evidence of no further significant loss of VMN between 24 h and 1 month after SCI (15). Quantification of glia. Glial nuclei counts were performed in a 0.2-mm 2 area of ventromedial white matter stained with hematoxylin. This area was bordered by the ventralmost edge of the tissue and the ventral sulcus. Previous histological examination of this area after acute and chronic SCI using this model showed that this region demonstrated the greatest and most consistent chronic sparing of white matter (25, 32, 37). By 15 min after SCI, total white matter nuclei were significantly reduced at the lesion epicenter and at distances of up to ⫾4 mm distal from the epicenter (Fig. 6A). At the 15-min time point, this represented a 20 –25% loss of total glial cells in the ventromedial white matter. We did not observe a significant additional loss of total glia at 4, 8, and 24 h after SCI (Fig. 6A). Slides representative of the lesion epicenter and extending out to 3 mm rostral and caudal were immunohistochemically labeled with either GFAP or CC1 (Figs. 6B and 6C). Counts of CC1- and GFAP-labeled cells performed within the same tissue area used for nuclei counts revealed that 50% of the total nuclei counted were oligodendrocytes and astrocytes as determined by immunolabeling. Only cells that were intensely immunolabeled were counted. Pathological
cells tended to stain faintly or had a granular appearance and were not included. The cells that were not immunohistochemically labeled were presumed to be microglia, endothelial cells, and white blood cells that infiltrated the area at the time of injury. At 15 min postinjury, the number of oligodendrocytes was significantly decreased at the epicenter compared to controls (Fig. 6B). A further loss of oligodendrocytes was observed at 4 h after SCI, a reduction of 50% compared to controls, which persisted at 8 and 24 h postinjury. At ⫾2 mm from the epicenter, the numbers of oligodendrocytes were significantly reduced compared to controls, with the loss at 15 min postinjury similar to that seen at 4, 8, and 24 h after SCI. Although the number of oligodendrocytes also tended to be reduced at ⫾3 mm compared to epicenter, the variability was such that the cell number was not statistically significant from controls (Fig. 6B). In contrast to oligodendrocytes, the number of astrocytes was not different from controls at 15 min after SCI at the lesion epicenter (Fig. 6C) and distances up to ⫾3 mm. By 4 h after SCI, astrocytes were significantly reduced by 60% at the lesion epicenter and reduced by 30% at distances of 2 and 3 mm from the epicenter (Fig. 6C). At 8 and 24 h after injury, astrocyte loss at the epicenter was similar to that observed at 4 h after SCI. At ⫾2 mm from the epicenter, we found that while
FIG. 5. Ventral motor neurons die in both a time- and a locationdependent manner after SCI. The number of viable VMN per horn was quantified in rats sacrificed at 15 min or 4, 8, or 24 h after SCI and in normal controls. VMN survival increased as a function of distance from the lesion epicenter. Negative numbers on the y axis represent mm distances rostral to epicenter, while positive numbers represent distances caudal to the epicenter. N ⫽ 5 rats per group. Every point below the dotted line is statistically different from control. *Significant difference compared to preceding time points. Statistical significance was determined with repeated-measures ANOVA followed by Tukey post hoc comparison with P ⱕ 0.05.
279
NEURONAL AND GLIAL DEATH AFTER SCI
FIG. 7. VMN and glial cell death over time. Graph represents average numbers of surviving cells at the lesion epicenters as a percentage of control at 15 min and 4, 8, and 24 h after SCI. Circles represent VMN, triangles represent oligodendrocytes, and squares represent astrocytes surviving after SCI. Where no error bars are seen, the SE was smaller than the symbol.
astrocyte numbers were significantly reduced compared to normal controls, or counts at 15 min postinjury, there were no significant differences comparing 4, 8, and 24 h. At ⫾3 mm, astrocyte numbers appeared significantly reduced at 4 and 8 h after SCI. However, by 24 h, astrocyte numbers were not significantly different from normal controls or the 15-min postinjury group (Fig. 6C). Comparison of neuronal and glial loss. A comparison of glial and VMN loss at the lesion epicenter over time showed that the pattern of cell loss was similar between oligodendrocytes and VMN (Fig. 7). Both were significantly decreased at 15 min after SCI and demonstrated maximum cell loss at 4 h postinjury. The 4-h postinjury time point is also important since this is when we see a severe decline in the number of astrocytes, whose numbers were not significantly affected at 15 min. Like oligodendrocytes and VMN, maximum astrocytic cell loss occurred at 4 h after SCI with no further loss observed at 8 or 24 h postinjury. Additional comparison of glial and VMN loss at ⫾3 mm from the lesion epicenter found that by 24 h after SCI only about 30% of VMN remained compared to an insignificant loss of astrocytes and oligodendrocytes (Figs. 5 and 6). DISCUSSION FIG. 6. Time course of glial loss after SCI. (A) Counts of total nuclei in the ventromedial white matter after SCI. Hematoxylin-stained sections were quantified at epicenter and distances up to 4 mm rostral and caudal to it at 15 min and 4, 8, and 24 h after SCI, compared to normal controls. Total glial nuclei were significantly decreased at all times and distances examined. (B) CC1-stained oligodendrocytes were quantified at 15 min and 4, 8, and 24 h after SCI. Oligodendrocytes were significantly decreased at epicenter, 1 mm, and 2 mm at all time points examined but there was no significance difference between injury groups and normal controls at 3 mm from the epicenter. (C) GFAPstained astrocytes were decreased after SCI at 4, 8, and 24 h at epicenter, 1 mm, and 2 mm, but at 15 min no decrease was observed. *Significant compared to normal control group. ␣Significant compared to the 15-min injury group. Significant compared to the 24-h injury group. Bars represent means ⫾ SE.
This study examined the temporal–spatial pattern of acute neuron and glial cell loss after experimental contusive SCI, focusing on VMN and glial cells in adjacent ventromedial white matter. The major findings of the current study are: (1) Cell loss expands in a symmetrical manner rostral and caudal to the injury epicenter over time. (2) While VMN and glia are both lost after SCI, significant VMN loss is more spatially extensive than glial loss (i.e., occurs at greater distances from the impact site). (3) While acute VMN loss seems attributable to necrosis, glial loss is associated with both necrosis and apoptosis. (4) VMN and oligodendrocytes exhibit the same temporal pattern of loss, indicating
280
GROSSMAN, ROSENBERG, AND WRATHALL
that they may be vulnerable to a common factor in the secondary injury cascade. (5) Astrocytes are the most resistant to SCI, showing the greatest degree of survival of the cell types examined, and exhibiting the longest time before the onset of cell loss. We have thus characterized the acute loss of three important classes of cells in the spinal cord after injury, with respect to both time and distance. This is important for further investigations of alterations that precede cell death and may thus be causally involved. Temporal–spatial pattern of cell loss. The loss of VMN acutely after SCI expanded symmetrically both rostrally and caudally far beyond the actual mechanical impact site, while glial loss was more restricted to areas of direct impact. VMN loss was also extensive, with only 44% of VMN still present at 4 mm distal from epicenter at 24 h after injury and none at the epicenter or at ⫾1 mm. Oligodendrocytes in ventral white matter demonstrated a similar temporal pattern but their loss was not as extensive. About 50% of oligodendrocytes were spared at the epicenter at 24 h, about 70% at ⫾2 mm, and no significant loss was seen at ⫾3 mm. Astrocytes were lost more slowly than oligodendrocytes with no significant loss until 4 h after injury. However, by 24 h only about 40% remained at the epicenter, 60% at ⫾2 mm, while their numbers were similar to normal at ⫾3 mm. Thus, significant glial loss extended over less distance and the acute secondary injury processes spared greater numbers of glial cells than neurons. Necrosis versus apoptosis? Our results also suggest a difference in the mechanisms of cell death in neurons, at least VMN, and glia in the first 24 h after SCI. Recently, apoptosis has been recognized as an important contributor to cell death during secondary injury after CNS trauma (1, 12, 18 –21, 24, 34, 35, 38). In the current study we found morphological evidence of extensive glial apoptosis, as well as examples of glial necrosis. However, we saw no evidence of apoptosis in VMN at the epicenter or at distances of up to 4 mm from it, at any time points examined. VMN pathology had morphological features of necrosis with loss of cell membrane integrity and cell swelling or condensation visible in plastic sections from tissue obtained at 4 and 24 h after SCI. Thus, although our results do not preclude the possibility of VMN apoptosis after SCI, they strongly suggest that it is not a major mechanism of cell death for these neurons in this model of SCI. In contrast, nearby glial cells frequently exhibit characteristics of apoptotic cell death. Our results are consistent with a number of previous reports that have shown apoptosis in glial cells both acutely and chronically after SCI, the latter often in oligodendrocytes associated with Wallerian degeneration (1, 12, 20, 21). Evidence for neuronal apoptosis after SCI is less clear. In three different studies, TUNEL staining of neurons was reported in the first
24 h after SCI in one study (21), was found to be restricted to dorsal horn neurons in the second (17), and was not observed in the third (19). The story is further complicated by the observation that only some TUNEL-positive cells seen after SCI actually show key features of apoptosis when examined by electron microscopy (24). Further, different populations of neurons may respond differently to injury, and SCI severity could also affect the extent of necrotic compared to apoptotic neuronal cell death. It is possible that VMN that we considered “lost” were simply shrunken due to metabolic alterations so that they were smaller than our size criteria but recovered at a later time. This is unlikely, since in a previous study in which we counted the number of viable VMN at ⫾4 mm, we found that the number at 24 h was the same as at 1 month after SCI (15). Similarly, others have reported that VMN are lost acutely in the first 1–2 days after SCI and this loss does not increase over time (17, 21). Our results suggest that treatments to spare VMN need to be administered in the first 24 h after injury. However, additional studies are needed to test his hypothesis. Our observation of necrotic cell death of VMN is consistent with glutamate-induced excitotoxicity (13, 16). In cerebellar and cortical cultures, excitotoxic cell death is not programmed (13, 16) but occurs by necrosis due to overwhelming influx of sodium and calcium (9). The acute necrotic loss of neurons may also be due to physical damage of either the perikarya or the processes. It is possible that VMN in the area of direct impact undergo such harsh mechanical injury that they lose the ability to carry out an apoptotic program (5). However, spinal cord ischemic injury in the rabbit (33) also showed delayed and selective death of motor neurons that was not associated with apoptosis. VMN and oligodendrocytes have the same temporal pattern of loss. Despite potential differences in mechanisms of cell death, acute loss of VMN and oligodendrocytes follows a similar pattern that is consistent with their susceptibility to glutamate-induced excitotoxicity (2, 39). After experimental SCI, neurons and oligodendrocytes exhibit caspase-3 activation (35). This happens early in neurons and hours to days later in oligodendrocytes adjacent to and distal to the injury site. Caspase-3 regulates the apoptotic cell death program (35). However, our evidence showing that oligodendrocyte numbers are reduced by 15 min after injury indicates that other modes of cell death are also involved. Astrocytes are lost more slowly. Astrocytes do not succumb to cell death to the same extent or as quickly as VMN and oligodendrocytes. Our results in contusive SCI are similar to those in a model of traumatic brain injury, in which more neurons than astrocytes died as a result of injury (24). Astrocytes are known to be resistant to glutamate toxicity, which could partially
NEURONAL AND GLIAL DEATH AFTER SCI
explain their resistance to injury. They have also been found, unlike neurons (22), to express high levels of the anti-apoptotic protein Bcl-2 (23) that may also render them resistant. The astrocyte death that is seen could be due to sensitivity to potassium toxicity (10), as potassium accumulates in the extracellular space after SCI (8). In order to improve therapy to reduce the effects of secondary injury after spinal cord trauma, it is critical to know which cells are most susceptible to death early after injury and when and where they are lost. It will also be important to find out why some cells are resistant while others are vulnerable. Importantly, we have identified populations of cells in specific locations after a standardized SCI that that can be used to study cellular alterations preceding cell loss in this model. How these data can be used is illustrated in the companion paper in which we examine changes in glutamate receptor subunits to determine whether they precede, and thus might be causally related to, loss of VMN after SCI.
9. 10.
11.
12.
13.
14.
15.
ACKNOWLEDGMENTS This study was supported by National Institutes of Health Grants RO1 NS 37733 and RO1 NS 35647 (J.R.W.) and NIMH NRSA 1F31 MH 12038 (S.D.G.).
16.
17.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
Abe, Y., T. Yamamoto, Y. Sugiyama, T. Watanabe, N. Saito, H. Kayama, and T. Kumagai. 1999. Apoptotic cells associated with Wallerian degeneration after experimental spinal cord injury: A possible mechanism of oligodendroglial death. J. Neurotrauma 16: 945–952. Annis, C. M., and J. E. Vaughn. 1998. Differential vulnerability of autonomic and somatic motor neurons to N-methyl-D-aspartate-induced excitotoxicity. Neuroscience 83: 239 –249. Basso, D. M., M. S. Beattie, and J. C. Bresnahan. 1996. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp. Neurol. 139: 244 –256. Bhat, R. V., K. J. Axt, J. S. Fosnaugh, K. J. Smith, K. A. Johnson, D. E. Hill, K. W. Kinzler, and J. M. Baraban. 1996. Expression of the APC tumor suppressor protein in oligodendroglia. Glia 17: 169 –174. Bonfoco, E., D. Krainc, M. Ankarcrona, P. Nicotera, and S. A. Lipton. 1995. Apoptosis and necrosis: Two distinct events induced, respectively, by mild and intense insults with N-methylD-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. USA 92: 7162–7166. Bresnahan, J. C., M. S. Beattie, B. T. Stokes, and K. M. Conway. 1991. Three-dimensional computer-assisted analysis of graded contusion lesions in the spinal cord of the rat. J. Neurotrauma 8: 91–101. Bresnahan, J. C., M. S. Beattie, F. D. d. Todd, and D. H. Noyes. 1987. A behavioral and anatomical analysis of spinal cord injury produced by a feedback-controlled impaction device. Exp. Neurol. 95: 548 –570. Chesler, M., W. Young, A. Z. Hassan, K. Sakatani, and T. Moriya. 1994. Elevation and clearance of extracellular K ⫹ following graded contusion of the rat spinal cord. Exp. Neurol. 125: 93–98.
18.
19.
20.
21.
22.
23.
24.
25.
26.
281
Choi, D. W. 1992. Excitotoxic cell death. J. Neurobiol. 23: 1261– 1276. Chow, S. Y., Y. C. Yen-Chow, H. S. White, L. Hertz, and D. M. Woodbury. 1991. Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons. Neurochem. Res. 16: 1275–1283. Coggeshall, R. E., and H. A. Lekan. 1996. Methods for determining numbers of cells and synapses: A case for more uniform standards of review. J. Comp. Neurol. 364: 6 –15. [Published erratum appears in J. Comp. Neurol., 1996, 369: 162] Crowe, M. J., J. C. Bresnahan, S. L. Shuman, J. N. Masters, and M. S. Beattie. 1997. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat. Med. 3: 73–76. [Published erratum appears in Nat. Med. 1997, 3: 240] Dessi, F., C. Charriaut-Marlangue, M. Khrestchatisky, and Y. Ben-Ari. 1993. Glutamate-induced neuronal death is not a programmed cell death in cerebellar culture. J. Neurochem. 60: 1953–1955. Gale, K., H. Kerasidis, and J. R. Wrathall. 1985. Spinal cord contusion in the rat: Behavioral analysis of functional neurologic impairment. Exp. Neurol. 88: 123–134. Grossman, S. D., B. B. Wolfe, R. P. Yasuda, and J. R. Wrathall. 2000. Changes in NMDA receptor subunit expression in response to contusive spinal cord injury. J. Neurochem. 75: 174 – 184. Gwag, B. J., J. Y. Koh, J. A. DeMaro, H. S. Ying, M. Jacquin, and D. W. Choi. 1997. Slowly triggered excitotoxicity occurs by necrosis in cortical cultures. Neuroscience 77: 393– 401. Kato, H., G. K. Kanellopoulos, S. Matsuo, Y. J. Wu, M. F. Jacquin, C. Y. Hsu, N. T. Kouchoukos, and D. W. Choi. 1997. Neuronal apoptosis and necrosis following spinal cord ischemia in the rat. Exp. Neurol. 148: 464 – 474. Katoh, K., T. Ikata, S. Katoh, Y. Hamada, K. Nakauchi, T. Sano, and M. Niwa. 1996. Induction and its spread of apoptosis in rat spinal cord after mechanical trauma. Neurosci. Lett. 216: 9 –12. Li, G. L., G. Brodin, M. Farooque, K. Funa, A. Holtz, W. L. Wang, and Y. Olsson. 1996. Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord. J. Neuropathol. Exp. Neurol. 55: 280 –289. Li, G. L., M. Farooque, A. Holtz, and Y. Olsson. 1999. Apoptosis of oligodendrocytes occurs for long distances away from the primary injury after compression trauma to rat spinal cord. Acta Neuropathol. (Berlin) 98: 473– 480. Liu, X. Z., X. M. Xu, R. Hu, C. Du, S. X. Zhang, J. W. McDonald, H. X. Dong, Y. J. Wu, G. S. Fan, M. F. Jacquin, C. Y. Hsu, and D. W. Choi. 1997. Neuronal and glial apoptosis after traumatic spinal cord injury. J. Neurosci. 17: 5395–5406. Lou, J., L. G. Lenke, F. J. Ludwig, and M. F. O’Brien. 1998. Apoptosis as a mechanism of neuronal cell death following acute experimental spinal cord injury. Spinal Cord 36: 683– 690. Mizuguchi, M., K. Ikeda, M. Asada, S. Mizutani, and S. Kamoshita. 1994. Expression of Bcl-2 protein in murine neural cells in culture. Brain Res. 649: 197–202. Newcomb, J. K., X. Zhao, B. R. Pike, and R. L. Hayes. 1999. Temporal profile of apoptotic-like changes in neurons and astrocytes following controlled cortical impact injury in the rat. Exp. Neurol. 158: 76 – 88. Noble, L. J., and J. R. Wrathall. 1985. Spinal cord contusion in the rat: Morphometric analyses of alterations in the spinal cord. Exp. Neurol. 88: 135–149. Noble, L. J., and J. R. Wrathall. 1989. Correlative analyses of lesion development and functional status after graded spinal cord contusive injuries in the rat. Exp. Neurol. 103: 34 – 40.
282 27.
28.
29.
30.
31.
32.
33.
34.
GROSSMAN, ROSENBERG, AND WRATHALL Panjabi, M. M., and J. R. Wrathall. 1988. Biomechanical analysis of experimental spinal cord injury and functional loss. Spine 13: 1365–1370. Rabchevsky, A. G., I. Fugaccia, A. Fletcher-Turner, D. A. Blades, M. P. Mattson, and S. W. Scheff. 1999. Basic fibroblast growth factor (bFGF) enhances tissue sparing and functional recovery following moderate spinal cord injury. J. Neurotrauma 16: 817– 830. Raines, A., K. L. Dretchen, K. Marx, and J. R. Wrathall. 1988. Spinal cord contusion in the rat: Somatosensory evoked potentials as a function of graded injury. J. Neurotrauma 5: 151–160. Rosenberg, L., Y. Teng, and J. Wrathall. 1999. 2,3-Dihydroxy6-nitro-7-sulfamoyl-benzo(f)quinoxaline reduces glial loss and acute white matter pathology after experimental spinal cord contusion. J. Neurosci. 19: 464 – 475. Rosenberg, L. J., Y. D. Teng, and J. R. Wrathall. 1999. Effects of the sodium channel blocker tetrodotoxin on acute white matter pathology after experimental contusive spinal cord injury. J. Neurosci. 19: 6122– 6133. Rosenberg, L. J., and J. R. Wrathall. 1997. Quantitative analysis of acute axonal pathology in experimental spinal cord contusion. J. Neurotrauma 14: 823– 838. Sakurai, M., T. Hayashi, K. Abe, M. Sadahiro, and K. Tabayashi. 1998. Delayed and selective motor neuron death after transient spinal cord ischemia: A role of apoptosis? J. Thoracic Cardiovasc. Surg. 115: 1310 –1315. Shuman, S. L., J. C. Bresnahan, and M. S. Beattie. 1997. Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats. J. Neurosci. Res. 50: 798 – 808.
35.
36.
37.
38.
39.
40.
41.
42.
Springer, J. E., R. D. Azbill, and P. E. Knapp. 1999. Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury. Nat. Med. 5: 943–946. Teng, Y. D., I. Mocchetti, A. M. Taveira-DaSilva, R. A. Gillis, and J. R. Wrathall. 1999. Basic fibroblast growth factor increases long-term survival of spinal motor neurons and improves respiratory function after experimental spinal cord injury. J. Neurosci. 19: 7037–7047. Teng, Y. D., and J. R. Wrathall. 1997. Local blockade of sodium channels by tetrodotoxin ameliorates tissue loss and long-term functional deficits resulting from experimental spinal cord injury. J. Neurosci. 17: 4359 – 4366. Wada, S., K. Yone, Y. Ishidou, T. Nagamine, S. Nakahara, T. Niiyama, and T. Sakou. 1999. Apoptosis following spinal cord injury in rats and preventative effect of N-methyl-D-aspartate receptor antagonist. J. Neurosurg. 91: 98 –104. Wetts, R., and J. E. Vaughn. 1996. Differential vulnerability of two subsets of spinal motor neurons in amyotrophic lateral sclerosis. Exp. Neurol. 141: 248 –255. Wrathall, J. R., D. Choiniere, and Y. D. Teng. 1994. Dosedependent reduction of tissue loss and functional impairment after spinal cord trauma with the AMPA/kainate antagonist NBQX. J. Neurosci. 14: 6598 – 6607. Wrathall, J. R., R. K. Pettegrew, and F. Harvey. 1985. Spinal cord contusion in the rat: Production of graded, reproducible, injury groups. Exp. Neurol. 88: 108 –122. Grossman, S. D., L. J. Rosenberg, and J. R. Wrathall. 2001. Relationship of altered glutamate receptor subunit mRNA expression to acute cell loss after spinal cord contusion. Exp. Neurol. 168: 283–289.
Temporal–Spatial Pattern of Acute Neuronal and Glial Loss after Spinal Cord Contusion S. D. Grossman, L. J. Rosenberg, and J. R. Wrathall Department of Cell Biology and Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road, Washington, DC 20007 Received April 11, 2000; accepted December 19, 2000
The secondary loss of neurons and glia over the first 24 h after spinal cord injury (SCI) contributes to the permanent functional deficits that are the unfortunate consequence of SCI. The progression of this acute secondary cell death in specific neuronal and glial populations has not previously been investigated in a quantitative manner. We used a well-characterized model of SCI to analyze the loss of ventral motoneurons (VMN) and ventral funicular astrocytes and oligodendrocytes at 15 min and 4, 8, and 24 h after an incomplete midthoracic contusion injury in the rat. We found that both the length of lesion and the length of spinal cord devoid of VMN increased in a time-dependent manner. The extent of VMN loss at specified distances rostral and caudal to the injury epicenter progressed symmetrically with time. Neuronal loss was accompanied by a loss of glial cells in ventral white matter that was significant at the epicenter by 4 h after injury. Oligodendrocyte loss followed the same temporal pattern as that of VMN while astrocyte loss was delayed. This information on the temporal–spatial pattern of cell loss can be used to investigate mechanisms involved in secondary injury of neurons and glia after SCI. © 2001 Academic Press Key Words: cell death; motoneurons; glia; electron microscopy; apoptosis; necrosis; GFAP; CC1; morphometry.
INTRODUCTION
In recent studies on experimental spinal cord injury (SCI), we observed that loss of large ventral horn motor neurons (VMN) adjacent to the thoracic injury site is extensive by 24 h after SCI with no evidence of significant additional cell loss of VMN 1 month later (15). The first 24 h after injury appears critical for loss of VMN. The loss of these neurons is functionally significant since their preservation through acute treatment with a neurotrophic factor results in improved respiratory function (36). We have also examined the development of white matter pathology and axonal loss that
occurs in ventromedial white matter in the first 24 h after SCI (32), which includes loss of glial cells. The oligodendrocyte loss at 24 h after injury can be significantly reduced by acute treatment with the AMPA/ kainate antagonist, NBQX (30), a treatment that results in increased chronic preservation of white matter and reduced long-term hind limb functional deficits (40). Thus, the loss of white matter glia in the first 24 h after SCI is also an important aspect of the secondary injury seen after spinal cord contusion. There have been a number of studies on histopathology after SCI that have used morphometric techniques to quantify tissue loss in gray and white matter (3, 6, 7, 25, 26). Recently a number of studies have focused on the occurrence of apoptosis in cells after SCI (1, 12, 21, 22, 38) and have provided quantitative information on the time course and location of cells demonstrating markers of apoptosis (21). However, to our knowledge there has been no previous quantitative study of the overall temporal–spatial pattern of loss of specific populations of cells after SCI. We therefore used a standardized and well-characterized rat model of incomplete SCI produced with a weight drop device (41) to assess the pattern of loss of VMN and ventromedial white matter glia at acute times after SCI. Loss of VMN, astrocytes, and oligodendrocytes at specific distances from the injury epicenter was quantified using a combination of immunohistochemistry and light and electron microscopy at 15 min, 4 h, 8 h, and 24 h after SCI. An accompanying study illustrates how these quantitative time-course data can be used to evaluate a potential mechanism of cell death involving alterations in glutamate receptor subunit expression (42). MATERIALS AND METHODS
Spinal cord injury. SCI was performed as previously described (15, 41). A total of 56 female, Sprague– Dawley rats weighing 200 –250 g were used for the studies. Animals were housed on a 12-h light/dark cycle with standard rat chow and water ad libitum.
273
0014-4886/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
274
GROSSMAN, ROSENBERG, AND WRATHALL
Rats were anesthetized with chloral hydrate (360 mg/kg ip), and a laminectomy was performed at T8, exposing the underlying dura. The spinal column was stabilized with angled Allis clamps on the T7 and T9 spinous processes. The impounder tip of a weight drop device (2.4 mm in diameter) was lowered onto the dura and a 10-g weight was dropped from a height of 2.5 cm onto the impounder to produce an incomplete spinal contusion. Controls for these experiments were age-, sex-, and weight-matched laminectomized rats. This model has been characterized in terms of biomechanics, resulting functional deficits, somatosensory evoked potentials, and quantitative histopathology (14, 25–27, 29). Histopathology. Spinal cords were rapidly removed from control and SCI rats (n ⫽ 5 at each time point) and 1.5-mm segments of thoracic cord, centered on the injury site, were quick frozen on dry ice and embedded in OCT (Tissue-Tek, Torrance, CA). Spinal cord segments were embedded in sets consisting of one or two segments from injured animals and one segment from an uninjured control and were sectioned at 20 m on a Jung Frigocut 2800 E cryostat. Sections were thaw mounted onto Superfrost Plus slides (Fisher) such that each slide represented 100 m of tissue and stored at ⫺20°C until used. Slides representative of each millimeter length of cord were stained with eriochrome RC (28), counterstained with hematoxylin and eosin, and used to reconstruct the injury site, as previously described (37). Lesion length was determined by tracing stained sections from each millimeter of spinal cord using a projector. We counted the numbers of sections exhibiting disruption of tissue and/or presence of hemorrhage to get a final lesion length in millimeters. Slides corresponding to the lesion epicenter and specific distances rostral and caudal to the epicenter were used in determining cell loss. Criteria for determination and quantification of ventral motoneurons. VMN were identified by their presence in the lower ventral horn, below the deep apex where the ventromedial gray and white matter meet, about 140 m below the central canal. Cells above this line were excluded. Counts were also based on size criteria. Specifically, VMN were counted by central profile counts (11) with use of a reticule that was divided into squares measuring 0.02 ⫻ 0.02 mm. Only VMN with diameter larger than half of one square were counted, with those counted having diameters ranging from 30 to 70 m. Inclusion also required the presence of a prominent nucleolus to avoid counting the same cell twice. Three slides were selected for each millimeter distance from the epicenter, each slide having 5 sections of spinal cord, for a total of 15 sections from each animal. The numbers of VMN per horn were counted in 10 sections, selected at random, according to
central profile counting procedures (11), and the average per horn was calculated. Immunohistochemical identification and quantification of glia. Slides adjacent to those used for VMN counts were labeled with antibody to glial fibrillary acid protein (GFAP; DAKO Corp., Carpinteria, CA) to identify astrocytes or CC1 (APC-7; Oncogene Research Products, Cambridge, MA) antibody, which recognizes the adenomatous polyposis coli gene expressed in oligodendrocytes (4, 12, 34). Primary antibodies were used at a dilution of 1:100 for GFAP and 1:1000 for CC1. CC1 used at this concentration is selective for identifying oligodendrocytes as determined by double immunolabeling with astrocyte and other oligodendrocyte antigens (4, 34). Endogenous peroxidase was quenched with 0.3% hydrogen peroxide in 0.1 M Tris buffer (pH 7.4) for 20 min. Sections were blocked for 1 h with 5% bovine serum albumin in 0.1 M Tris buffer. The blocking serum was removed and the tissue was exposed overnight at 4°C to the primary antibodies. The next day, the tissue was washed with Tris buffer and exposed to the secondary antibody for 30 – 45 min. Labeling was visualized using the ABC peroxidase technique (Vector Laboratories, Inc., Burlingame, CA) with 3,3⬘-diaminobenzidine or the VIP substrate kit (Vector Laboratories, Inc.). Sections were counterstained with hematoxylin or methyl green (Vector) for the purpose of determining total nuclei present. Counts of labeled glial cells were performed in a 0.2-mm 2 area of ventromedial white matter. Two slides representative of the lesion epicenter and specific millimeter distances rostral and caudal to it were examined. The first and fifth section from the five sections on each slide were used for quantification purposes. Only intensely labeled cells with a clearly defined nucleus were used to establish numbers of astrocytes and oligodendrocytes. Hematoxylin counts were performed at the same time to determine numbers of total nuclei. Electron microscopy. At 4 and 24 h after SCI, rats (n ⫽ 5 per group) were anesthetized with 4% chloral hydrate and intracardially perfused with saline followed by 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4. The spinal cords were removed from the vertebral column and fixed overnight in fresh fixative. A 2-cm segment, centered on the epicenter of the injury, was cut from each cord and embedded in 4% agar. The embedded tissue blocks were mounted on a tissue chopper stage (Sorvall, Newtown, CT) and cut into 250-m cross sections (4 sections ⫽ 1 mm of tissue). Tissue sections from the epicenter and from 1 mm rostral and caudal to the epicenter were used in studies of acute axonal pathology (30, 31). For the current study, sections at 2 and 4 mm rostral and caudal to the epicenter were postfixed for 1 h in 1% osmium–1% potassium ferricyanide, en bloc stained with 1% uranyl acetate and flat-embedded in
NEURONAL AND GLIAL DEATH AFTER SCI
Spurr resin (Ted Pella, Inc., Redding, CA). One-micrometer sections were cut from each tissue block, stained with 1% toluidine blue, and viewed with light microscopy. Blocks were trimmed to approximately 1 mm 2 in areas of interest and ultrathin (70 –90 nm) sections were cut and placed on 200-mesh nickel grids (Ted Pella, Inc.). Sections were viewed with a JEOL Jem 1200 EX (Tokyo, Japan) transmission electron microscope. Statistical analysis. The averages for each rat at each location were used to calculate means and standard errors for the group (n ⫽ 5 rats) at each time point examined. Statistical significance was determined using SigmaStat, a computerized statistics program (SPSS, San Raphael, CA). A one- or two-factor analysis of variance (ANOVA) followed by a post hoc test (Tukey) was performed with P values set at 0.05 to establish statistical differences between specific groups or time points. RESULTS
We examined spinal cord tissue from animals sacrificed 15 min or 4, 8, or 24 h after SCI or laminectomy. In spinal cords from laminectomy controls, we saw tightly packed cells in the gray matter with healthy appearing VMN (Fig. 1A). Gray matter was clearly delineated from white matter that contained structurally intact glia and axons. At 15 min after injury at the epicenter, many VMN were lost and the tight network of cells seen in controls was abolished (Fig. 1B). Distinction between gray and white matter was still apparent. At 4 and 8 h after SCI, there was virtual destruction of the ventral horns at the epicenters. At 8 h, white matter and gray matter were no longer clearly distinguishable due to tissue disruption and degeneration (Fig. 1C). At 24 h after injury, this disruption became even more apparent, and large areas of tissue were completely lost (Fig. 1D). Close examination of ventral horns showed there was complete loss of VMN by 24 h at the epicenter. This progressive destruction of the cord following SCI was seen not only at the lesion epicenter but also rostral and caudal to it. At 15 min tissue disruption appeared localized mainly in the area of direct mechanical impact (epicenter ⫾ 1 mm) with hemorrhage, primarily in dorsal funicular white matter, extending farther rostral and caudal to the impact site. At 2 mm from the epicenter, viable VMN and intact ventral white matter were still present. Tissue disruption was evident only in central gray and dorsal white matter, leaving the majority of the cord morphologically intact. The lesion expanded radially and longitudinally by 4 and 8 h, with further destruction of gray matter and mild to moderate disruption of white matter adjacent to the gray matter. By 24 h (Fig. 1E) a large cavity was formed at the epicenter, surrounded by a rim of intact
275
peripheral white matter. At distances of ⫾2 mm from epicenter, gray matter was still present, though clearly altered by the injury. Viable VMN were rarely seen. While visibly disrupted, a considerable amount of white matter remained. At 4 mm rostral and caudal, tissue damage was localized to the dorsal funicular white matter (Fig. 1E). At this location the gray matter appeared remarkably intact 24 h after SCI with a number of viable VMN seen, and white matter displayed little, if any, damage by light microscopy. Examination of residual ventral white matter at 24 h at the epicenter showed numerous hematoxylinstained glial bodies with abnormal nuclei characterized by nuclear condensation and fragmentation (Fig. 1G). Normal glial nuclei in laminectomy controls appeared as small, round structures with a smooth, intact edge (Fig. 1F). In order to visualize entire cells and to distinguish between subtypes of glia, immunolabeling was performed. Normal oligodendrocytes stained with CC1 displayed large cell bodies, dark and evenly stained cytoplasm (Fig. 1H). Lightly stained processes could be seen radiating short distances from the cell body. Oligodendrocytes at 24 h after SCI lacked radiating processes and displayed punctate dark aggregates in the cytoplasm and nucleus (Fig. 1I). Astrocytes labeled with GFAP also demonstrated striking morphological changes at 24 h after SCI (Fig. 1K). Normal astrocytes had small, intensely labeled cell bodies and numerous long processes extending from the perikaryon cell (Fig. 1J). After injury, these processes appeared discontinuous and the perikarya had rounded (Fig. 1K). Labeling intensity in the cell bodies was greatly diminished but astrocyte processes displayed increased immunoreactivity To better evaluate morphological changes occurring in VMN and glia after injury, 1-m plastic sections of spinal cord tissue were examined 4 and 24 h after SCI at distances of 2 and 4 mm from the epicenter. Neuronal pathology observed was consistently indicative of necrotic cell death, with no evidence of apoptosis seen at the time points examined. Neurons showed extensive and variable pathology (Figs. 2A–2D). Many of the neurons looked pyknotic, with vacuolization either in the nucleus or in the cytoplasm (Figs. 2A, 2B, and 2D). Others neurons appeared swollen and faintly stained, resulting in a “ghostly” appearance (Fig. 2C). The nuclei of the moribund VMN ranged from a large, swollen appearance with intact nuclear envelope (Fig. 2C) to a dark, condensed structure. Further, nucleoli also exhibited a variety of pathologies. While some nucleoli looked small and condensed (Fig. 2C), others appeared swollen and large (Fig. 2B), while others were fragmented (Fig. 2D). Although most of the neuronal pathology we observed was more consistent with a necrotic, rather than an apoptotic, phenotype, this was not the case in glia (Fig. 3). In general, most glia in the ventromedial
276
GROSSMAN, ROSENBERG, AND WRATHALL
FIG. 1. Microscopic examination of tissue pathology after SCI. Bright-field micrographs of spinal cord ventral horn at 20⫻ magnification showing a normal ventral horn (A) and ventral horns at 15 min (B), 8 h (C), and 24 h after SCI (D). Arrows show VMN included for cell counts, fitting size criteria and displaying a nucleolus. Arrowhead points to VMN excluded from analysis. Scale bar, 0.175 mm. (E) Photographs displaying a typical spinal cord lesion at 24 h after injury. The center section represents lesion epicenter. Sections left and right of the center section represent spinal cord sections at 2 mm rostral and caudal to the epicenter. Sections to the far right and left are taken at distances of 4 mm rostral and caudal to the epicenter. Scale bar, 0.5 mm. (F–K) Glial pathology at 24 h after SCI at the epicenter compared to normal controls. Ventromedial white matter stained with hematoxylin was used to visualize normal (F) and pathological (G) nuclei at 24 h after SCI. Oligodendrocytes or astrocytes were identified with the antibodies CC1 and GFAP. Photographs represent typical pathological changes seen in oligodendrocytes (I) and astrocytes (K) at 24 h after SCI at the lesion epicenter, compared to their respective controls (H and J).
white matter after SCI appeared swollen. In many, the nuclear chromatin appeared in dispersed clumps throughout the nucleus. In other cells, the nuclei were
irregularly shaped, with a single condensed aggregate of chromatin. Glial apoptosis was confirmed by electron microscopy at 24 h after SCI (Fig. 3C).
NEURONAL AND GLIAL DEATH AFTER SCI
277
FIG. 2. Selected ventral motor neurons show various pathologies after SCI. Moribund VMN were photographed at 100⫻ magnification at 2 mm from the epicenter at 4 h after SCI (A, C, D) or at 4 mm from the epicenter at 24 h after SCI (B). Extensive and variable pathology can be seen in 1-m plastic sections of ventral gray matter stained with toluidine blue.
Quantification of neurons. Having observed a progressive spread of injury rostral and caudal to the impact site, slides representing sequential millimeter distances distal to the epicenter were selected for quantitative lesion analysis at 15 min or 4, 8, or 24 h after SCI (Fig. 4). At 15 min the average length of the lesions (defined as presence of tissue disruption and/or hemorrhage) was about 9 mm. There was a progressive increase in lesion length over time. By 24 h the lesion length averaged about 13 mm, representing a 30% increase from 15 min postinjury (Fig. 4). There was also a progressive loss of residual VMN. Control rats had an average of approximately 10 VMN per horn (based on criteria described under Materials and Methods) at all thoracic locations examined (Fig. 5). At 15 min after SCI, only 33% of the normal number of VMN were present at the epicenter of injury. At ⫾1 mm from the epicenter 48% of control VMN were still present. At ⫾2 and 3 mm, however, approximately 50 and 60% of VMN remained, respectively. By ⫾4 mm, however, the numbers of VMN in injured rats was not statistically different from control numbers, averaging 91% of control values. VMN counts in rats sacrificed 4 and 8 h after SCI (Fig. 5) showed no difference between the two experimental groups, although they were significantly different from controls at all distances examined (Fig. 5). Complete VMN loss was seen at epicenter. At 4 mm
FIG. 3. Glial pathology as seen by microscopic examination of ventromedial white matter. (A) Swollen glia were typically found at 4 h after SCI. Nuclei demonstrated a grainy appearance (arrowhead) or contained clumped chromatin (arrow). Generally, the nuclear envelope was intact. (B) Many glia had irregular nuclei and were dark in appearance (arrowheads). Typically, the nuclear envelope of these cells was rough or ragged in appearance as opposed to the smooth nuclear envelope seen in glia with swollen nuclei (A). (C) High-magnification (2000⫻) showed that a number of glia had an apoptotic phenotype at 24 h after SCI as demonstrated by the presence of compartmentalized, chromatin bodies within the cytoplasm.
278
GROSSMAN, ROSENBERG, AND WRATHALL
FIG. 4. Lesion length increases with time after SCI. Graph represents quantification of the length of spinal cord lesion in rats sacrificed 15 min or 4, 8, or 24 h after SCI. The length of lesion increases over time after the injury, to reach significantly longer lengths at 24 h compared to lesion length observed at 15 min postinjury. N ⫽ 5 rats per group. Bars represent means ⫾ SE. *Significant difference compared to 15-min time point. Statistical significance was determined with a Tukey post hoc test with P ⬍ 0.05.
distal, injured rats had 77% of control numbers of VMN. By 4 and 8 h, there was approximately a 2.5- to 3-mm segment of cord lacking VMN. By 24 h, tissue destruction was so severe that VMN were completely lost at the injury epicenter and 2 mm in either direction. The survival rate at 4 mm rostral and caudal was approximately 44% and the length of cord devoid of VMN measured 4 mm (Fig. 5). At each time point we examined, the loss of VMN was symmetrical rostral and caudal to the injury epicenter. It is important to note that in this model there is evidence of no further significant loss of VMN between 24 h and 1 month after SCI (15). Quantification of glia. Glial nuclei counts were performed in a 0.2-mm 2 area of ventromedial white matter stained with hematoxylin. This area was bordered by the ventralmost edge of the tissue and the ventral sulcus. Previous histological examination of this area after acute and chronic SCI using this model showed that this region demonstrated the greatest and most consistent chronic sparing of white matter (25, 32, 37). By 15 min after SCI, total white matter nuclei were significantly reduced at the lesion epicenter and at distances of up to ⫾4 mm distal from the epicenter (Fig. 6A). At the 15-min time point, this represented a 20 –25% loss of total glial cells in the ventromedial white matter. We did not observe a significant additional loss of total glia at 4, 8, and 24 h after SCI (Fig. 6A). Slides representative of the lesion epicenter and extending out to 3 mm rostral and caudal were immunohistochemically labeled with either GFAP or CC1 (Figs. 6B and 6C). Counts of CC1- and GFAP-labeled cells performed within the same tissue area used for nuclei counts revealed that 50% of the total nuclei counted were oligodendrocytes and astrocytes as determined by immunolabeling. Only cells that were intensely immunolabeled were counted. Pathological
cells tended to stain faintly or had a granular appearance and were not included. The cells that were not immunohistochemically labeled were presumed to be microglia, endothelial cells, and white blood cells that infiltrated the area at the time of injury. At 15 min postinjury, the number of oligodendrocytes was significantly decreased at the epicenter compared to controls (Fig. 6B). A further loss of oligodendrocytes was observed at 4 h after SCI, a reduction of 50% compared to controls, which persisted at 8 and 24 h postinjury. At ⫾2 mm from the epicenter, the numbers of oligodendrocytes were significantly reduced compared to controls, with the loss at 15 min postinjury similar to that seen at 4, 8, and 24 h after SCI. Although the number of oligodendrocytes also tended to be reduced at ⫾3 mm compared to epicenter, the variability was such that the cell number was not statistically significant from controls (Fig. 6B). In contrast to oligodendrocytes, the number of astrocytes was not different from controls at 15 min after SCI at the lesion epicenter (Fig. 6C) and distances up to ⫾3 mm. By 4 h after SCI, astrocytes were significantly reduced by 60% at the lesion epicenter and reduced by 30% at distances of 2 and 3 mm from the epicenter (Fig. 6C). At 8 and 24 h after injury, astrocyte loss at the epicenter was similar to that observed at 4 h after SCI. At ⫾2 mm from the epicenter, we found that while
FIG. 5. Ventral motor neurons die in both a time- and a locationdependent manner after SCI. The number of viable VMN per horn was quantified in rats sacrificed at 15 min or 4, 8, or 24 h after SCI and in normal controls. VMN survival increased as a function of distance from the lesion epicenter. Negative numbers on the y axis represent mm distances rostral to epicenter, while positive numbers represent distances caudal to the epicenter. N ⫽ 5 rats per group. Every point below the dotted line is statistically different from control. *Significant difference compared to preceding time points. Statistical significance was determined with repeated-measures ANOVA followed by Tukey post hoc comparison with P ⱕ 0.05.
279
NEURONAL AND GLIAL DEATH AFTER SCI
FIG. 7. VMN and glial cell death over time. Graph represents average numbers of surviving cells at the lesion epicenters as a percentage of control at 15 min and 4, 8, and 24 h after SCI. Circles represent VMN, triangles represent oligodendrocytes, and squares represent astrocytes surviving after SCI. Where no error bars are seen, the SE was smaller than the symbol.
astrocyte numbers were significantly reduced compared to normal controls, or counts at 15 min postinjury, there were no significant differences comparing 4, 8, and 24 h. At ⫾3 mm, astrocyte numbers appeared significantly reduced at 4 and 8 h after SCI. However, by 24 h, astrocyte numbers were not significantly different from normal controls or the 15-min postinjury group (Fig. 6C). Comparison of neuronal and glial loss. A comparison of glial and VMN loss at the lesion epicenter over time showed that the pattern of cell loss was similar between oligodendrocytes and VMN (Fig. 7). Both were significantly decreased at 15 min after SCI and demonstrated maximum cell loss at 4 h postinjury. The 4-h postinjury time point is also important since this is when we see a severe decline in the number of astrocytes, whose numbers were not significantly affected at 15 min. Like oligodendrocytes and VMN, maximum astrocytic cell loss occurred at 4 h after SCI with no further loss observed at 8 or 24 h postinjury. Additional comparison of glial and VMN loss at ⫾3 mm from the lesion epicenter found that by 24 h after SCI only about 30% of VMN remained compared to an insignificant loss of astrocytes and oligodendrocytes (Figs. 5 and 6). DISCUSSION FIG. 6. Time course of glial loss after SCI. (A) Counts of total nuclei in the ventromedial white matter after SCI. Hematoxylin-stained sections were quantified at epicenter and distances up to 4 mm rostral and caudal to it at 15 min and 4, 8, and 24 h after SCI, compared to normal controls. Total glial nuclei were significantly decreased at all times and distances examined. (B) CC1-stained oligodendrocytes were quantified at 15 min and 4, 8, and 24 h after SCI. Oligodendrocytes were significantly decreased at epicenter, 1 mm, and 2 mm at all time points examined but there was no significance difference between injury groups and normal controls at 3 mm from the epicenter. (C) GFAPstained astrocytes were decreased after SCI at 4, 8, and 24 h at epicenter, 1 mm, and 2 mm, but at 15 min no decrease was observed. *Significant compared to normal control group. ␣Significant compared to the 15-min injury group. Significant compared to the 24-h injury group. Bars represent means ⫾ SE.
This study examined the temporal–spatial pattern of acute neuron and glial cell loss after experimental contusive SCI, focusing on VMN and glial cells in adjacent ventromedial white matter. The major findings of the current study are: (1) Cell loss expands in a symmetrical manner rostral and caudal to the injury epicenter over time. (2) While VMN and glia are both lost after SCI, significant VMN loss is more spatially extensive than glial loss (i.e., occurs at greater distances from the impact site). (3) While acute VMN loss seems attributable to necrosis, glial loss is associated with both necrosis and apoptosis. (4) VMN and oligodendrocytes exhibit the same temporal pattern of loss, indicating
280
GROSSMAN, ROSENBERG, AND WRATHALL
that they may be vulnerable to a common factor in the secondary injury cascade. (5) Astrocytes are the most resistant to SCI, showing the greatest degree of survival of the cell types examined, and exhibiting the longest time before the onset of cell loss. We have thus characterized the acute loss of three important classes of cells in the spinal cord after injury, with respect to both time and distance. This is important for further investigations of alterations that precede cell death and may thus be causally involved. Temporal–spatial pattern of cell loss. The loss of VMN acutely after SCI expanded symmetrically both rostrally and caudally far beyond the actual mechanical impact site, while glial loss was more restricted to areas of direct impact. VMN loss was also extensive, with only 44% of VMN still present at 4 mm distal from epicenter at 24 h after injury and none at the epicenter or at ⫾1 mm. Oligodendrocytes in ventral white matter demonstrated a similar temporal pattern but their loss was not as extensive. About 50% of oligodendrocytes were spared at the epicenter at 24 h, about 70% at ⫾2 mm, and no significant loss was seen at ⫾3 mm. Astrocytes were lost more slowly than oligodendrocytes with no significant loss until 4 h after injury. However, by 24 h only about 40% remained at the epicenter, 60% at ⫾2 mm, while their numbers were similar to normal at ⫾3 mm. Thus, significant glial loss extended over less distance and the acute secondary injury processes spared greater numbers of glial cells than neurons. Necrosis versus apoptosis? Our results also suggest a difference in the mechanisms of cell death in neurons, at least VMN, and glia in the first 24 h after SCI. Recently, apoptosis has been recognized as an important contributor to cell death during secondary injury after CNS trauma (1, 12, 18 –21, 24, 34, 35, 38). In the current study we found morphological evidence of extensive glial apoptosis, as well as examples of glial necrosis. However, we saw no evidence of apoptosis in VMN at the epicenter or at distances of up to 4 mm from it, at any time points examined. VMN pathology had morphological features of necrosis with loss of cell membrane integrity and cell swelling or condensation visible in plastic sections from tissue obtained at 4 and 24 h after SCI. Thus, although our results do not preclude the possibility of VMN apoptosis after SCI, they strongly suggest that it is not a major mechanism of cell death for these neurons in this model of SCI. In contrast, nearby glial cells frequently exhibit characteristics of apoptotic cell death. Our results are consistent with a number of previous reports that have shown apoptosis in glial cells both acutely and chronically after SCI, the latter often in oligodendrocytes associated with Wallerian degeneration (1, 12, 20, 21). Evidence for neuronal apoptosis after SCI is less clear. In three different studies, TUNEL staining of neurons was reported in the first
24 h after SCI in one study (21), was found to be restricted to dorsal horn neurons in the second (17), and was not observed in the third (19). The story is further complicated by the observation that only some TUNEL-positive cells seen after SCI actually show key features of apoptosis when examined by electron microscopy (24). Further, different populations of neurons may respond differently to injury, and SCI severity could also affect the extent of necrotic compared to apoptotic neuronal cell death. It is possible that VMN that we considered “lost” were simply shrunken due to metabolic alterations so that they were smaller than our size criteria but recovered at a later time. This is unlikely, since in a previous study in which we counted the number of viable VMN at ⫾4 mm, we found that the number at 24 h was the same as at 1 month after SCI (15). Similarly, others have reported that VMN are lost acutely in the first 1–2 days after SCI and this loss does not increase over time (17, 21). Our results suggest that treatments to spare VMN need to be administered in the first 24 h after injury. However, additional studies are needed to test his hypothesis. Our observation of necrotic cell death of VMN is consistent with glutamate-induced excitotoxicity (13, 16). In cerebellar and cortical cultures, excitotoxic cell death is not programmed (13, 16) but occurs by necrosis due to overwhelming influx of sodium and calcium (9). The acute necrotic loss of neurons may also be due to physical damage of either the perikarya or the processes. It is possible that VMN in the area of direct impact undergo such harsh mechanical injury that they lose the ability to carry out an apoptotic program (5). However, spinal cord ischemic injury in the rabbit (33) also showed delayed and selective death of motor neurons that was not associated with apoptosis. VMN and oligodendrocytes have the same temporal pattern of loss. Despite potential differences in mechanisms of cell death, acute loss of VMN and oligodendrocytes follows a similar pattern that is consistent with their susceptibility to glutamate-induced excitotoxicity (2, 39). After experimental SCI, neurons and oligodendrocytes exhibit caspase-3 activation (35). This happens early in neurons and hours to days later in oligodendrocytes adjacent to and distal to the injury site. Caspase-3 regulates the apoptotic cell death program (35). However, our evidence showing that oligodendrocyte numbers are reduced by 15 min after injury indicates that other modes of cell death are also involved. Astrocytes are lost more slowly. Astrocytes do not succumb to cell death to the same extent or as quickly as VMN and oligodendrocytes. Our results in contusive SCI are similar to those in a model of traumatic brain injury, in which more neurons than astrocytes died as a result of injury (24). Astrocytes are known to be resistant to glutamate toxicity, which could partially
NEURONAL AND GLIAL DEATH AFTER SCI
explain their resistance to injury. They have also been found, unlike neurons (22), to express high levels of the anti-apoptotic protein Bcl-2 (23) that may also render them resistant. The astrocyte death that is seen could be due to sensitivity to potassium toxicity (10), as potassium accumulates in the extracellular space after SCI (8). In order to improve therapy to reduce the effects of secondary injury after spinal cord trauma, it is critical to know which cells are most susceptible to death early after injury and when and where they are lost. It will also be important to find out why some cells are resistant while others are vulnerable. Importantly, we have identified populations of cells in specific locations after a standardized SCI that that can be used to study cellular alterations preceding cell loss in this model. How these data can be used is illustrated in the companion paper in which we examine changes in glutamate receptor subunits to determine whether they precede, and thus might be causally related to, loss of VMN after SCI.
9. 10.
11.
12.
13.
14.
15.
ACKNOWLEDGMENTS This study was supported by National Institutes of Health Grants RO1 NS 37733 and RO1 NS 35647 (J.R.W.) and NIMH NRSA 1F31 MH 12038 (S.D.G.).
16.
17.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
Abe, Y., T. Yamamoto, Y. Sugiyama, T. Watanabe, N. Saito, H. Kayama, and T. Kumagai. 1999. Apoptotic cells associated with Wallerian degeneration after experimental spinal cord injury: A possible mechanism of oligodendroglial death. J. Neurotrauma 16: 945–952. Annis, C. M., and J. E. Vaughn. 1998. Differential vulnerability of autonomic and somatic motor neurons to N-methyl-D-aspartate-induced excitotoxicity. Neuroscience 83: 239 –249. Basso, D. M., M. S. Beattie, and J. C. Bresnahan. 1996. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp. Neurol. 139: 244 –256. Bhat, R. V., K. J. Axt, J. S. Fosnaugh, K. J. Smith, K. A. Johnson, D. E. Hill, K. W. Kinzler, and J. M. Baraban. 1996. Expression of the APC tumor suppressor protein in oligodendroglia. Glia 17: 169 –174. Bonfoco, E., D. Krainc, M. Ankarcrona, P. Nicotera, and S. A. Lipton. 1995. Apoptosis and necrosis: Two distinct events induced, respectively, by mild and intense insults with N-methylD-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. USA 92: 7162–7166. Bresnahan, J. C., M. S. Beattie, B. T. Stokes, and K. M. Conway. 1991. Three-dimensional computer-assisted analysis of graded contusion lesions in the spinal cord of the rat. J. Neurotrauma 8: 91–101. Bresnahan, J. C., M. S. Beattie, F. D. d. Todd, and D. H. Noyes. 1987. A behavioral and anatomical analysis of spinal cord injury produced by a feedback-controlled impaction device. Exp. Neurol. 95: 548 –570. Chesler, M., W. Young, A. Z. Hassan, K. Sakatani, and T. Moriya. 1994. Elevation and clearance of extracellular K ⫹ following graded contusion of the rat spinal cord. Exp. Neurol. 125: 93–98.
18.
19.
20.
21.
22.
23.
24.
25.
26.
281
Choi, D. W. 1992. Excitotoxic cell death. J. Neurobiol. 23: 1261– 1276. Chow, S. Y., Y. C. Yen-Chow, H. S. White, L. Hertz, and D. M. Woodbury. 1991. Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons. Neurochem. Res. 16: 1275–1283. Coggeshall, R. E., and H. A. Lekan. 1996. Methods for determining numbers of cells and synapses: A case for more uniform standards of review. J. Comp. Neurol. 364: 6 –15. [Published erratum appears in J. Comp. Neurol., 1996, 369: 162] Crowe, M. J., J. C. Bresnahan, S. L. Shuman, J. N. Masters, and M. S. Beattie. 1997. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat. Med. 3: 73–76. [Published erratum appears in Nat. Med. 1997, 3: 240] Dessi, F., C. Charriaut-Marlangue, M. Khrestchatisky, and Y. Ben-Ari. 1993. Glutamate-induced neuronal death is not a programmed cell death in cerebellar culture. J. Neurochem. 60: 1953–1955. Gale, K., H. Kerasidis, and J. R. Wrathall. 1985. Spinal cord contusion in the rat: Behavioral analysis of functional neurologic impairment. Exp. Neurol. 88: 123–134. Grossman, S. D., B. B. Wolfe, R. P. Yasuda, and J. R. Wrathall. 2000. Changes in NMDA receptor subunit expression in response to contusive spinal cord injury. J. Neurochem. 75: 174 – 184. Gwag, B. J., J. Y. Koh, J. A. DeMaro, H. S. Ying, M. Jacquin, and D. W. Choi. 1997. Slowly triggered excitotoxicity occurs by necrosis in cortical cultures. Neuroscience 77: 393– 401. Kato, H., G. K. Kanellopoulos, S. Matsuo, Y. J. Wu, M. F. Jacquin, C. Y. Hsu, N. T. Kouchoukos, and D. W. Choi. 1997. Neuronal apoptosis and necrosis following spinal cord ischemia in the rat. Exp. Neurol. 148: 464 – 474. Katoh, K., T. Ikata, S. Katoh, Y. Hamada, K. Nakauchi, T. Sano, and M. Niwa. 1996. Induction and its spread of apoptosis in rat spinal cord after mechanical trauma. Neurosci. Lett. 216: 9 –12. Li, G. L., G. Brodin, M. Farooque, K. Funa, A. Holtz, W. L. Wang, and Y. Olsson. 1996. Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord. J. Neuropathol. Exp. Neurol. 55: 280 –289. Li, G. L., M. Farooque, A. Holtz, and Y. Olsson. 1999. Apoptosis of oligodendrocytes occurs for long distances away from the primary injury after compression trauma to rat spinal cord. Acta Neuropathol. (Berlin) 98: 473– 480. Liu, X. Z., X. M. Xu, R. Hu, C. Du, S. X. Zhang, J. W. McDonald, H. X. Dong, Y. J. Wu, G. S. Fan, M. F. Jacquin, C. Y. Hsu, and D. W. Choi. 1997. Neuronal and glial apoptosis after traumatic spinal cord injury. J. Neurosci. 17: 5395–5406. Lou, J., L. G. Lenke, F. J. Ludwig, and M. F. O’Brien. 1998. Apoptosis as a mechanism of neuronal cell death following acute experimental spinal cord injury. Spinal Cord 36: 683– 690. Mizuguchi, M., K. Ikeda, M. Asada, S. Mizutani, and S. Kamoshita. 1994. Expression of Bcl-2 protein in murine neural cells in culture. Brain Res. 649: 197–202. Newcomb, J. K., X. Zhao, B. R. Pike, and R. L. Hayes. 1999. Temporal profile of apoptotic-like changes in neurons and astrocytes following controlled cortical impact injury in the rat. Exp. Neurol. 158: 76 – 88. Noble, L. J., and J. R. Wrathall. 1985. Spinal cord contusion in the rat: Morphometric analyses of alterations in the spinal cord. Exp. Neurol. 88: 135–149. Noble, L. J., and J. R. Wrathall. 1989. Correlative analyses of lesion development and functional status after graded spinal cord contusive injuries in the rat. Exp. Neurol. 103: 34 – 40.
282 27.
28.
29.
30.
31.
32.
33.
34.
GROSSMAN, ROSENBERG, AND WRATHALL Panjabi, M. M., and J. R. Wrathall. 1988. Biomechanical analysis of experimental spinal cord injury and functional loss. Spine 13: 1365–1370. Rabchevsky, A. G., I. Fugaccia, A. Fletcher-Turner, D. A. Blades, M. P. Mattson, and S. W. Scheff. 1999. Basic fibroblast growth factor (bFGF) enhances tissue sparing and functional recovery following moderate spinal cord injury. J. Neurotrauma 16: 817– 830. Raines, A., K. L. Dretchen, K. Marx, and J. R. Wrathall. 1988. Spinal cord contusion in the rat: Somatosensory evoked potentials as a function of graded injury. J. Neurotrauma 5: 151–160. Rosenberg, L., Y. Teng, and J. Wrathall. 1999. 2,3-Dihydroxy6-nitro-7-sulfamoyl-benzo(f)quinoxaline reduces glial loss and acute white matter pathology after experimental spinal cord contusion. J. Neurosci. 19: 464 – 475. Rosenberg, L. J., Y. D. Teng, and J. R. Wrathall. 1999. Effects of the sodium channel blocker tetrodotoxin on acute white matter pathology after experimental contusive spinal cord injury. J. Neurosci. 19: 6122– 6133. Rosenberg, L. J., and J. R. Wrathall. 1997. Quantitative analysis of acute axonal pathology in experimental spinal cord contusion. J. Neurotrauma 14: 823– 838. Sakurai, M., T. Hayashi, K. Abe, M. Sadahiro, and K. Tabayashi. 1998. Delayed and selective motor neuron death after transient spinal cord ischemia: A role of apoptosis? J. Thoracic Cardiovasc. Surg. 115: 1310 –1315. Shuman, S. L., J. C. Bresnahan, and M. S. Beattie. 1997. Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats. J. Neurosci. Res. 50: 798 – 808.
35.
36.
37.
38.
39.
40.
41.
42.
Springer, J. E., R. D. Azbill, and P. E. Knapp. 1999. Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury. Nat. Med. 5: 943–946. Teng, Y. D., I. Mocchetti, A. M. Taveira-DaSilva, R. A. Gillis, and J. R. Wrathall. 1999. Basic fibroblast growth factor increases long-term survival of spinal motor neurons and improves respiratory function after experimental spinal cord injury. J. Neurosci. 19: 7037–7047. Teng, Y. D., and J. R. Wrathall. 1997. Local blockade of sodium channels by tetrodotoxin ameliorates tissue loss and long-term functional deficits resulting from experimental spinal cord injury. J. Neurosci. 17: 4359 – 4366. Wada, S., K. Yone, Y. Ishidou, T. Nagamine, S. Nakahara, T. Niiyama, and T. Sakou. 1999. Apoptosis following spinal cord injury in rats and preventative effect of N-methyl-D-aspartate receptor antagonist. J. Neurosurg. 91: 98 –104. Wetts, R., and J. E. Vaughn. 1996. Differential vulnerability of two subsets of spinal motor neurons in amyotrophic lateral sclerosis. Exp. Neurol. 141: 248 –255. Wrathall, J. R., D. Choiniere, and Y. D. Teng. 1994. Dosedependent reduction of tissue loss and functional impairment after spinal cord trauma with the AMPA/kainate antagonist NBQX. J. Neurosci. 14: 6598 – 6607. Wrathall, J. R., R. K. Pettegrew, and F. Harvey. 1985. Spinal cord contusion in the rat: Production of graded, reproducible, injury groups. Exp. Neurol. 88: 108 –122. Grossman, S. D., L. J. Rosenberg, and J. R. Wrathall. 2001. Relationship of altered glutamate receptor subunit mRNA expression to acute cell loss after spinal cord contusion. Exp. Neurol. 168: 283–289.