A Mapping Study of Caspase-3 Activation Following Acute Spinal Cord Contusion in Rats
Department of Physical Medicine and Rehabilitation, University of Kentucky Medical Center, Spinal Cord and Brain Injury Research Center, and Cardinal Hill Rehabilitation Hospital, Lexington, Kentucky
Correspondence to: Melanie L. McEwen, Ph.D., Department of Physical Medicine and Rehabilitation, University of Kentucky Medical Center, Cardinal Hill Rehabilitation Hospital, 800 Rose St., MN 225, Lexington, KY 40536-0298. E-mail: mlmcew2{at}uky.edu
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Summary |
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Key Words: apoptosis contusion myelin secondary injury spinal cord injury central nervous system trauma
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Introduction |
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Although our understanding of apoptotic cell death following SCI is far from complete, much is now known about individual intracellular proteins with apoptotic or anti-apoptotic functions. Evidence suggests that the morphological and nuclear changes that occur during apoptosis are initiated and executed through activation of the evolutionarily conserved caspase family of cysteine proteases (Thornberry 1997; Thornberry and Lazebnik 1998
). Recent findings have established that caspase-3 is activated in humans (Emery et al. 1998
; Harter et al. 2001
; Hentze et al. 2001
) and in animal models (Yakovlev et al. 1997
; Springer et al. 1999
; Beer et al. 2000
; Citron et al. 2000
; Clark et al. 2000
; Casha et al. 2001
; Nottingham et al. 2002
; Sullivan et al. 2002
) following traumatic brain and SCI. Although activated caspase-3 has been localized to a variety of cell types post-SCI (Springer et al. 1999
; Citron et al. 2000
; Clark et al. 2000
; Beattie et al. 2002
) and was correlated with DNA fragmentation (Casha et al. 2001
), the time course of caspase-3 activation has not been thoroughly examined.
The purpose of the present experiment was to perform a descriptive analysis of the spatiotemporal distribution of activated caspase-3 at acute times following SCI (up to 8 days postinjury), and to determine if activated caspase-3 colocalized with cellular markers for neurons, oligodendroglia, or microglia/macrophages. Because there is clear evidence for the existence of caspase-3independent apoptotic cell death pathways (Li et al. 2001; van Loo et al. 2001
; Cande et al. 2002
; Arnoult et al. 2003
), this study was conducted to elucidate the time course of caspase-3 activation in the acute periods following SCI. The results of this study serve as a springboard for quantifying the effectiveness of therapeutic interventions that limit the activation or activity of caspase-3.
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Materials and Methods |
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Surgery
All surgical techniques were performed under aseptic conditions. Rats were anesthetized with 40 mg/kg sodium pentobarbital (Nembutal; Abbott Laboratories; North Chicago, IL) administered intraperitoneally and dorsal incisions were made in the skin and underlying muscles. The muscles were retracted and a laminectomy was performed at thoracic (T) segment 10 (T10). The muscles of control rats (sham surgery) were closed with absorbable sutures, and the skin was closed with wound clips. Sham control rats were then placed into an incubator (35C) for 2 to 3 hr to recover from anesthesia. The vertebral column of the remaining rats was stabilized by clamping the vertebrae at T8 and T11. The spinal cord was subsequently injured by dropping a 10-g rod, with a tip that measured 2.5 mm in diameter, a distance of 12.5 mm onto the exposed dorsal surface of the spinal cord (NYU Impactor device; New York University, New York, NY). The incision site of those rats was closed in layers as described and the rats were allowed to recover from anesthesia. All rats were then returned to their home cages in the colony room until euthanized. Bladders of the spinally injured rats were manually expressed 2x per day until micturition was reestablished or until the rats were euthanized.
Histology
After the appropriate survival period, the rats were deeply anesthetized with sodium pentobarbital (100 mg/kg) and perfused transcardially with 50 ml of 0.1 M phosphate-buffered saline (PBS), followed by 200 ml of 4% paraformaldehyde in PBS (pH, 7.27.4). A 2-cm segment of spinal cord centered on the lesion/laminectomy site was removed and placed into 25% sucrose-PBS at 4C for 5 days. Spinal cords were then rapidly frozen on dry ice and stored at 20C until they were embedded in tissue freezing medium (Triangle Biomedical Sciences; Durham, NC) and cut into 30-µm-thick transverse sections on a cryostat. Consecutive spinal cord sections were mounted in pairs on a series of charged glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA) so that adjacent pairs on each slide represented regions
1 mm (990 µm) apart in the spinal cord. After the sections air dried, the slides were stored at 4C until they were stained for myelin and Nissl substance or until fluorescence immunocytochemistry was performed.
Tissue sections from each subject were stained for myelin and Nissl substance for brightfield microscopy as follows: briefly, sections were dehydrated at room temperature (RT) in a graded ethanol series (5 min each) and cleared in Hemo-De (Fisher Scientific) for 5 min. Sections were then rehydrated in a reverse-graded ethanol series, followed by distilled water (5 min each). To stain myelinated fibers, the tissue sections were placed for 10 min into a solution that contained 0.16% Eriochrome cyanine-R (Sigma; St Louis, MO), 0.4% sulfuric acid, 0.4% iron chloride, and 0.12% hydrochloric acid and then rinsed under running tap water for 10 min. Sections were then differentiated in 1% ammonium hydroxide for 4 min and again rinsed under running tap water for 10 min. To stain neurons, the sections were placed in 0.1 M acetate buffer for 1 min and then stained in 0.03% neutral red (Sigma) in 0.1 M acetate buffer for 15 min. After staining, the slides were rinsed in distilled water (3 x 5 min), dehydrated in a graded ethanol series (2 min each), cleared in Hemo-De (2 x 2 min) (Fisher Scientific) and coverslipped with Permount (Fisher Scientific).
Adjacent slides were processed for fluorescence immunocytochemistry as follows: briefly, sections were rinsed for 1 hr at RT in several changes of PBS. Sections were then preincubated for 30 min at RT in a blocking solution comprised of 5% normal goat serum (NGS) and 0.05% Triton X-100 in PBS. The blocking solution was removed and sections were incubated in a humidified chamber overnight at 4C in PBS that contained 5% NGS and one of the following sets of primary antibodies: (a) rabbit anti-human/mouse activated caspase-3 IgG (polyclonal, 1:4000; R & D Systems, Minneapolis, MN) and mouse anti-APC/Ab7 (CC1) IgG (monoclonal, 1:250; Oncogene, Cambridge, MA), (b) anti-activated caspase-3 and mouse anti-neuronal nuclei (NeuN) IgG (monoclonal, 1:250; Chemicon International, Temecula, CA), or (c) anti-activated caspase-3 and mouse anti-rat CD11b/c (OX42) IgG (monoclonal, 1:4000; PharMingen, San Diego, CA). To test for nonspecific staining by the secondary antibodies, additional slides were processed in a similar fashion with the primary antibodies excluded. All slides were then rinsed for 1 hr at RT in several changes of PBS and incubated in the dark for 1 hr at RT in PBS that contained 5% NGS and the following fluorescent secondary antibodies: Cy-3-conjugated goat anti-rabbit IgG (polyclonal, 1:500; Jackson ImmunoResearch Laboratories, West Grove, PA) and AlexaFluor 488 goat anti-mouse IgG (monoclonal, 1:250; Molecular Probes, Eugene, OR). The secondary antibody solution was removed and the slides were rinsed in several changes of PBS for 15 min at RT. Tissue sections were immediately coverslipped with ProLong Anti-Fade (Molecular Probes) and stored at 4C in the dark to retard fading of the fluorescent labels.
Tissue sections were subsequently examined using a Zeiss AxioPlan microscope (Zeiss; Oberkochen, Germany). Myelin and Nissl staining were examined under brightfield, and images were captured with a Zeiss AxioCam color digital camera (x50). Colocalization of the fluorescent secondary antibodies was determined when paired images of each field were captured (x200 or x400) and superimposed using Photoshop (Adobe Systems; San Jose, CA). Immunoreactive cells were identified in gray (dorsal and ventral horn) and white matter. The analysis of immunoreactive cells in white matter was restricted primarily to the ventral funiculus, which contains descending supraspinal motor pathways affected by SCI in the rat.
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Results |
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Discussion |
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Although caspase-3 activation may not necessarily indicate cells that will eventually die by apoptosis (Zeuner et al. 1999; Nicotera et al. 2000
; Racke et al. 2002
), it is noteworthy that the time-course of caspase-3 activation following spinal cord contusion reported here parallels previous reports of DNA fragmentation, as identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) or Hoechst staining (Crowe et al. 1997
; Liu et al. 1997
; Shuman et al. 1997
). However, the potential contribution of a caspase-3independent apoptotic process cannot be overlooked and, therefore, our findings underscore the importance of correlating caspase-3 activation with other markers of apoptosis.
Activated caspase-3 in the contused spinal cord was primarily colocalized with CC1-positive cells, a marker for oligodendroglia (Bhat et al. 1996). Interestingly, the time-dependent decrease and resurgence of activated caspase-3 following SCI paralleled a time-dependent loss and reemergence of CC1-positive oligodendroglia in the spinal cord white matter within 2 mm of the lesion epicenter. Specifically, the initial upregulation of activated caspase-3 at 4 hr and 1 day postinjury was accompanied by a subsequent decrease in the number of CC1-positive oligodendroglia. Both cellular markers were sparse for the next several days postinjury. On postoperative day 8, activated caspase-3 was again present when CC1-positive cells were again abundant near the lesion epicenter. At this time-point, colocalization of activated caspase-3 and CC1 was again observed (see also Nottingham et al. 2002
). Recent studies have also demonstrated a time-dependent loss and reemergence of oligodendroglia following SCI (Frei et al. 2000
; McTigue et al. 2001
). Previous reports have indicated that the number of mature oligodendroglia of the spinal cord dramatically decreases 34 days postinjury, especially near the lesion epicenter (McTigue et al. 2001
). Several recent reports have suggested that oligodendroglial progenitor cells of the spinal cord increase after SCI (Ishii et al. 2001
; McTigue et al. 2001
; Jones et al. 2002
; Jones and Tuszynski 2002
). Death of mature oligodendroglia and subsequent proliferation and maturation of oligodendroglial progenitors may explain the observed loss and reemergence of CC1-positive cells 8 days post-SCI. However, injury-related changes in antigen expression or antigen binding by CC1 are possible alternative interpretations, which has been reported for neuronal markers following cerebral ischemia (Unal-Cevik et al. 2004
).
A few NeuN-positive neurons of the dorsal horn were also immunoreactive for activated caspase-3, primarily at 4 hr and 1 day postcontusion. Activated caspase-3 and NeuN were primarily colocalized to the small neurons of the dorsal horn. In contrast, NeuN-positive motoneurons of the ventral horn were not immunoreactive for activated caspase-3 at any time-point examined. Although previous reports have suggested that motoneurons of the adult spinal cord undergo apoptosis post-SCI (Yong et al. 1998), which may be mediated by caspase-3 (Citron et al. 2000
), experimental differences in injury severity or in the time-points examined may explain the different outcomes.
We found that only a few OX42-positive cells appeared to be immunoreactive for activated caspase-3. When present, activated caspase-3 was most prevalent at 4 hr postinjury in OX42-positive cells that had a ramified morphology and shortened processes, the morphological characteristics of partially activated microglia/macrophages (Popovich et al. 1997; Shuman et al. 1997
; Carlson et al. 1998
). Our findings correspond with previous reports on spinal cords that received less severe contusions in which macrophages with apoptotic bodies were occasionally noted (Liu et al. 1997
), and with previous descriptions of spinal cords that received more severe contusions where microglia/macrophage apoptosis was widespread (Shuman et al. 1997
; Yong et al. 1998
). Because macrophages can recognize and engulf cells undergoing apoptosis (Duvall et al. 1985
; Savill et al. 1993
), colocalization of OX42 and activated caspase-3 may indicate macrophages that devoured cells in which caspase-3 was previously activated and not necessarily indicate that the OX42-positive cells themselves expressed the activated form of this cell death protein (Shuman et al. 1997
).
Although there is evidence of astrocyte cell death following SCI (Grossman et al. 2001), and subpopulations of astrocytes in dorsal horn of the uninjured spinal cord appear to express activated caspase-3 (Noyan-Ashraf et al. 2005
), we have not found conclusive evidence of activated caspase-3 in astrocytes following mild to moderate SCI (Nottingham S and Springer J, unpublished data; however, see Yong et al. 1998
). This may be due in part to the staining pattern of the astrocyte marker glial fibrillary acidic protein (GFAP). It is difficult to differentiate GFAP-positive cell bodies containing activated caspase-3 from GFAP-positive astrocytic processes encircling a caspase-3positive cell (Nottingham S and Springer J, unpublished data). Although confocal microscopy might be more revealing, labeling of GFAP and activated caspase-3 was not pursued in the present study due to the extremely rare occurrence of potentially double-labeled cells. However, this observation does raise an important point with respect to the role of reactive astrocytes in formation of the glial scar, which is known to limit the potential for functional regeneration or sprouting. Specifically, the relative absence of caspase-3 colocalization in GFAP-positive cells following SCI might suggest that reactive astrocytes may be relatively more resistant to cell death signals in the injured spinal cord. As a consequence, a lack of apoptotic signaling may contribute to promote formation of the growth-inhibiting glial scar.
Cells that contained activated caspase-3 were present in the spinal cords of rats that received sham surgery and may reflect cellular responses to nonspecific trauma from the laminectomy procedure (e.g., hypoxia, edema). It is important to stress that the temporal distribution of activated caspase-3 within the spinal cords of sham controls was vastly different from the distribution of activated caspase-3 within the injured spinal cords. Following sham surgery, activated caspase-3 was observed in cells of the gray and white matter at all postsurgical time-points examined. In contrast, cells that contained activated caspase-3 in the injured spinal cord dramatically decreased and was sparse on postoperative days 2 and 4 in all subjects that received a spinal cord contusion. These data suggest that the cellular mechanisms activated in response to a "mild stressor" (sham surgery) or to a rapid traumatic impact to the spinal cord may differentially regulate caspase-3 activation.
Overall, the results of this experiment indicate that during acute times following SCI, caspase-3 activation occurs primarily in oligodendroglia in areas of relatively intact white matter. Some small neurons of the dorsal horn were immunopositive for activated caspase-3 postinjury, whereas neurons of the ventral horn were not immunopositive for activated caspase-3 under the conditions of the present experiment. These findings suggest that caspase-3mediated apoptosis is differentially regulated in neurons, which may reveal neuronal differences in the time-course of caspase-3mediated cell death or in the biochemical mediators of apoptosis. Finally, activated caspase-3 was rarely localized to OX42-positive microglia/macrophages and was most abundant at 4 hr postinjury. Caspase activation can alter cell structure (Kothakota et al. 1997; Rudel and Bokoch 1997
), so the early localization of activated caspase-3 to OX42-positive cells that appeared to have shortened processes may indicate a role for activated caspase-3 in the morphological transition of microglia/macrophages from a non-reactive to an activated phagocytic phenotype.
In conclusion, the results of this study are the first to report the pattern and cellular localization of activated caspase-3 in the injured spinal cord. These findings are particularly relevant given the fact that not all apoptotic cell death is caspase-3 dependent (Li et al. 2001; van Loo et al. 2001
; Cande et al. 2002
; Arnoult et al. 2003
). Relatively little is known about the survival signals important to mature oligodendroglia or to newly differentiating oligodendroglial progenitors of the adult nervous system, and whether those factors are still present or re-expressed following SCI. Therefore, the development of strategies that inhibit factors responsible for initiating and executing oligodendroglial apoptosis postinjury will be critical for promoting oligodendroglial survival and, hopefully, functional recovery.
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Acknowledgments |
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Footnotes |
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