Activation of Complement Pathways after Contusion

JOURNAL OF NEUROTRAUMA Volume 21, Number 12, 2004 © Mary Ann Liebert, Inc. Pp. 1831–1846

Activation of Complement Pathways after Contusion-Induced Spinal Cord Injury AILEEN J. ANDERSON,1 STEPHANIE ROBERT,2 WENCHENG HUANG,3 WISE YOUNG,3 and CARL W. COTMAN2

ABSTRACT Previous studies have shown that a cellular inflammatory response is initiated, and inflammatory cytokines are synthesized, following experimental spinal cord injury (SCI). In the present study, we tested the hypothesis that the complement cascade, a major component of both the innate and adaptive immune response, is also activated following experimental SCI. We investigated the pathways, cellular localization, timecourse, and degree of complement activation in rat spinal cord following acute contusion-induced SCI using the New York University (NYU) weight drop impactor. Mild and severe injuries (12.5 and 50 mm drop heights) at 1, 7, and 42 days post injury time points were evaluated. Classical (C1q and C4), alternative (Factor B) and terminal (C5b-9) complement pathways were strongly activated within 1 day of SCI. Complement protein immunoreactivity was predominantly found in cell types vulnerable to degeneration, neurons and oligodendrocytes, and was not generally observed in inflammatory or astroglial cells. Surprisingly, immunoreactivity for complement proteins was also evident 6 weeks after injury, and complement activation was observed as far as 20 mm rostral to the site of injury. Axonal staining by C1q and Factor B was also observed, suggesting a potential role for the complement cascade in demyelination or axonal degeneration. These data support the hypothesis that complement activation plays a role in SCI. Key words: complement cascade; demyelination; immune response; inflammation; neuron; neurodegeneration; oligodendrocyte

INTRODUCTION

P

have shown that a cellular inflammatory response is initiated, and inflammatory cytokines are synthesized, following dorsal hemisection (Bartholdi and Schwab, 1997; Dusart and Schwab, 1994) or contusion-induced spinal cord injury (SCI) (Carlson et REVIOUS STUDIES

al., 1998; Popovich et al., 1997; Streit et al., 1998). Infiltration of inflammatory cells and corresponding cytokine production has been predicted to contribute to secondary injury via several mechanisms, and studies have suggested that inhibition of inflammation can be beneficial to recovery. High dose methylprednisolone (MP) therapy in humans provides a small but significant im-

1Departments

of Physical Medicine and Rehabilitation, Anatomy and Neurobiology, and the Reeve-Irvine Center, University of California, Irvine, California. 2Institute for Brain Aging and Dementia, University of California, Irvine, California. 3Neuroscience Center, Rutgers State University of New Jersey, Piscataway, New Jersey.

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ANDERSON ET AL. provement in functional outcome after SCI in clinical trials (Bracken et al., 2000). Recent studies have demonstrated that MP profoundly inhibits polymorphonuclear granulocyes and macrophage/microglia infiltration (Bartholdi and Schwab, 1995; Oudega et al., 1999), inhibits spinal tissue loss, and promotes regenerative sprouting (Chen et al., 1996; Kanellopoulos et al., 1997; Naso et al., 1995; Oudega et al., 1999), after hemisection- or transection-induced SCI in rodents. Similarly, studies investigating other inhibitors of inflammation, including depletion or suppression of mononuclear phagocytes (Blight, 1994; Giulian and Robertson, 1990; Popovich et al., 1999; Tonai et al., 2001), inhibition of leukocyte activation (Taoka and Okajima, 1998), inhibition of inflammatory angiogenesis (Wamil et al., 1998), administration of classical immunosuppressives (Bavetta et al., 1999), administration of anti-inflammatory cytokines (Bethea et al., 1999; Brewer et al., 1999), or direct inhibition of cytokine/chemokine activation (Ghirnikar et al., 2000; Nesic et al., 2001), have demonstrated that inhibition of inflammation after SCI is beneficial. Conversely, other work has suggested that stimulation of the inflammatory response via administration of autoimmune T-cells, implantation of stimulated homologous macrophages, or vaccination with Nogo-A-derived or myelin peptides can promote functional recovery after SCI (Hauben et al., 2001; Hauben et al., 2001; Hauben et al., 2000; Rapalino et al., 1998; Schwartz and Hauben, 2002). Thus, the role of inflammation in degeneration and regeneration after traumatic SCI remains unclear. Critically, in this regard, little attention has been paid to one of the principal effectors of these inflammatory events, the complement cascade. Complement is composed of over 30 cellular and plasma/serum proteins and is critical for homologous (host) defense. Complement deficiency results in a dramatic susceptibility to bacterial infection and increased incidence of immune complex illnesses such as systemic lupus erythematosus. Conversely, complement activation has been implicated in numerous CNS conditions, including traumatic brain injury (Hicks et al., 2002; Kossmann et al., 1997; Rancan et al., 2003; Stahel et al., 2000; Stahel et al., 1998; Stahel et al., 2001), and neurological disorders such as myasthenia gravis, Guillain-Barre syndrome, Alzheimer’s disease, and multiple sclerosis (MS), where complement activation is thought to mediate demyelination and neurodegeneration (Eikelenboom and Veerhuis, 1996; Morgan, 1995). The pathophysiological consequences of complement activation, and therapeutic efficacy of complement inhibition, have been demonstrated in several paradigms, including the middle cerebral artery occlusion model of

stroke and brain ischemia (Barnum, 1999; Huang et al., 1999; Makrides, 1998). The complement system can be divided into two basic enzymatic cascades, the classical and alternative pathways of activation, which converge upon a common mechanism for the assembly of a lytic membrane complex, the terminal pathway. The classical pathway is generally associated with the antibody-dependent binding of C1q to the Fc portion of IgG via its globular head cluster. However, molecules including A
peptide, mannanbinding protein, C-reactive protein, serum amyloid P, and myelin can also activate the classical pathway, either through antibody-independent interactions with C1q or C1, or by substituting for these proteins in the cascade (Eggleton et al., 1998; Morgan, 1995). Similarly, Factor B (FB) binding to C3b deposited on the surface of cellular membranes induces the activation of the alternative complement pathway, which was the first antibody-independent mechanism of complement activation identified (Morgan, 1995). These pathways converge upon the terminal pathway and nonenzymatic insertion of a pore composed of C5b, C6, C7, C8, and C9 into the cellular membrane; this complex is termed C5b-9 or the membrane attack complex (MAC). C5b-9 complex pore size is directly dependent on the number of C9 molecules incorporated, thus, formation of the membrane attack complex can lead to a range of cellular consequences, from Ca2+ influx and alterations in signaling pathways to cell death (Morgan, 1989; Morgan, 1995). In the present study we examine the pathways, cellular localization, timecourse, and degree of complement activation in rat spinal cord following acute contusioninduced SCI. Our findings support the hypothesis that complement activation plays an important role in spinal cord injury.

MATERIALS AND METHODS Surgical Procedures and Tissue Preparation All experiments were conducted in accordance with Institutional Animal Care and Use Committee guidelines. Rats were weighed to obtain pre-injury body weight and anesthetized with pentobarbital. Hooded Long-Evans rats, 77
3 days old, were subjected to contusion injury using the NYU weight drop impactor as described previously (Constantini and Young, 1994). Mild (n  18) or severe (n  18) contusion injuries were induced using a 10-g weight dropped 12.5 or 50 mm, respectively, onto T9-10 spinal cord exposed by laminectomy. Experimental animals were sacrificed 1, 7, or 42 days post-injury. Control animals (n  6) received laminectomy only and

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SCI-INDUCED COMPLEMENT ACTIVATION were sacrificed 1 day later. At the time of sacrifice, rats were anesthetized with pentobarbital and perfused with 4% paraformaldehyde in phosphate buffered saline, pH 7.4. Dissected spinal cords were immersed in 20% sucrose/4% paraformaldehyde at 4°C for 24 h and frozen on dry ice. Spinal cords were divided into 5-mm sequential blocks for cryosectioning. Serial 20-m sections were collected onto Vectabond-coated (Vector Labs) slides. Following sectioning, slides were dried on a slide warmer at 45°C for 30 min, distributed into slide boxes with desiccant, and stored at 20°C as described previously (Guthrie et al., 1993). Heating above 45°C, or storage at room temperature, severely attenuated complement protein antigenicity. Slides were stored such that boxes were thawed only once, immediately before use, and slides from different parameters (e.g., 12.5 mm C1q at 1 day, 7 days, and 42 days) were evenly distributed across staining runs to minimize potential variations in staining.

Immunocytochemistry Immunostaining was conducted essentially as described previously (Anderson et al., 1996), with the following modifications. Specifically, a 1-day immunostaining protocol was designed to optimize retention and quality of slide-mounted sections. Room-temperature slides were immersed in plain Tris (100 mM Tris-HCl, pH 7.4), followed by inactivation of endogenous peroxidases with 3% H2O2/10% methanol for 10 min. Slides were then washed with Tris-A (Tris pH 7.4 with 0.1% Triton x-100) for 15 min, blocked with Tris-B (Tris-A with 2% BSA and 2% normal horse serum) for 15 min, exposed to primary antibody in Tris-B for 2 h, washed for 30 min each with Tris-A and Tris-B, exposed to secondary antibody in Tris-B for 1 h, washed for 30 min each with Tris-A and Tris-B, and exposed to ABC complex for 1 h (Vector Elite, Burlingame, CA). Incubation in primary antibodies, secondary antibodies, and ABC complex was performed in 200 l chamberwells (RPI) in order to optimize staining and minimize reagent use. All other treatments/wash steps were performed in 50mL Coplin jars. After ABC complex, slides were washed in Tris-A for a minimum of 30 minutes, rinsed twice in plain Tris, and reacted with diaminobenzidine (DAB) for 5 min to visualize immunoreactivity (Vector, Burlingame, CA). Complement protein primary antibodies were tested for cross-reactivity with rat complement proteins in preliminary experiments and final antibody selections made on the basis of these data. Antibodies used were as follows: Goat anti-human C1q (Quidel A301; 1:500), goat

anti-human C4 (Quidel A305; 1:500), goat anti-human FB (Quidel A311; 1:500), mouse anti-human C5b-9 (Quidel A239; 1:500). Anti-C5b-9 was raised against a purified human C9, and has been shown to bind a neoantigen expressed on C5b-9 complex (terminal complement complex, membrane attack complex), poly-C9, and denatured immobilized C9 (Quidel). Biotinylated secondary antibodies preadsorbed against rat IgGs were obtained from Jackson ImmunoResearch (Westgrove, PA; 1:500), and were used in all experiments to prevent nonspecific labeling in injured tissue. Omission of either primary or secondary antibodies at these titrations produced uniformly negative immunostaining. Anti-complement primary antibodies were directed against whole protein, no peptide antibodies cross-reactive with rat complement proteins were available. Preadsorbtion of anti-complement primary antibodies with whole complement proteins or peptide antigen (10-fold excess) dramatically attenuated cellular immunoreactivity. Double labeling was conducted using Cy3 (red)– and Cy2 (green)–conjugated streptavidin (Jackson ImmunoResearch, Westgrove, PA; 1:150) in combination with the primary and preadsorbed secondary antibodies described above. Blue fluorescent nuclear counterstaining was visualized using DAPI (4,6-diamidino-2phenylindole) mounting medium (Vector). Oligodendrocytes were immunolabeled using mouse anti-human adenomatous polyposis coli (APC) clone CC-1 tumor suppressor obtained from Oncogene Research (San Diego, CA; Ab-7 OP80, 1:20). Potential for CC-1 staining overlap with astrocytes was investigated using rabbit anti-cow glial fibrillary acidic protein (GFAP) obtained from DAKO (Carpinteria, CA; Z0334; 1:10,000). Neurons and axons were immunolabeled using mouse antihuman neurofilament protein antibodies obtained from Sternberger Monoclonals (Lutherville, MD): neurons (SMI 32; 1:20,000), axons (SMI 31; 1:15,000). Omission of either primary or secondary antibodies at these titrations produced negative immunostaining.

Quantification of Cell Numbers and Section Areas Morphological observations were made in all 36 SCI animals and six laminectomy controls. Quantification was carried out in four animals for each timepoint from the 12.5-mm contusion injury, 50-mm contusion injury, and laminectomy control groups. Sections from three regions of the spinal cord relative to the injury epicenter, 5–10 mm caudal, 5–10 mm rostral, and 15–20 mm rostral, were used for immunocytochemical labeling and analysis of cell number (a minimum of three sections per

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ANDERSON ET AL. region, 9 sections per spinal cord). The injury epicenter was identified on the basis of histological assessment and Luxol Fast Blue staining of alternate sections. For cell counting, spinal cord hemisections were photographed with Fujichrome 64T at an absolute magnification of 132, using a 4 objective and 3.3 eyepiece, in order to optimize observation of the sections. 35-mm slides of these hemisections were projected onto a tablet with one inch grids; each stained profile in the section was morphologically identified as neuronal, oligodendroglial, or axonal, indicated on the traced section, counted, and the resulting data compiled for analysis. In addition, total section area, gray matter section area, and white matter section area were obtained from hemisections corresponding to those analyzed for complement protein immunostaining. Using digitally captured images and NIH Image software, total hemisection and gray matter outlines were manually traced, and areas calculated in NIH Image using calibration of pixels/mm with a stage micrometer (microscope and camera configuration identical to that for analysis). Gray matter section area was subsequently subtracted from total section area to obtain white matter section area. Investigators were blinded to experimental groups for all stages of quantification. Statistical significance was determined by 1- or 2-way ANOVA and post-hoc Fisher protected least significant difference (PLSD) with a required p  0.05 (StatView, Abacus Software).

RESULTS Complement Pathway Activation Immunolabeling for C1q (classical pathway), C4 (classical pathway), FB (alternative pathway), or C5b-9 (terminal pathway) was not observed in uninjured laminectomy control spinal cord (Fig. 1A,C,E,G). In contrast, robust immunolabeling for C1q, C4, FB and C5b-9 was observed throughout the gray and white matter of contusion-injured spinal cord (Fig. 1B,D,F,H), at all injury severities (12.5 mm or 50 mm impact height) and lengths of time following injury (1 day, 7 days, or 42 days), reflecting a dramatic change in the numbers of immunoreactive cells. Complement proteins were observed both rostral and caudal to the site of impact injury. Qualitatively, immunolabeling rostral to the impact tended to be greater than that observed caudal to the injury. Surprisingly, immunoreactivity for all complement proteins examined was observed quite distant from the site of impact, over 20 mm rostral to the lesion epicenter. Moreover, the degree of cellular labeling did not appear to decline with increasing distance from the epicenter, suggesting that the magnitude of the complement response

was essentially the same at all regions of the spinal cord examined.

Morphological Characterization of Labeling SCI animals exhibited immunoreactivity for complement proteins in a variety of structures, including C1q, C4, FB, and C5b-9 immunolabeling of neurons in the dorsal and ventral horns, and oligodendroglial cells located in the dorsal, lateral, and ventral funiculi (Figs. 1 and 2). Neuronal staining was verified using SMI 32 (data not shown). In contrast with previous studies in which extracellular C1q and C9 staining has been reported in the neuropil after brain insult, and suggested to be associated with sites of synaptic loss and synaptogenesis (Johnson et al., 1996), we did not observe neuropil complement immunoreactivity in the present study. Morphologically, complement-immunoreactive cells in the white matter funiculi appeared to be oligodendrocytes; triple fluorescent labeling for complement proteins, the oligodendroglial marker CC-1, and DAPI supports this interpretation (Fig. 3). Low levels of CC-1-immunoreactivity have been reported in a subset of astrocytes (Bhat et al., 1996); however, in separate experiments double labeling for CC-1 and GFAP in spinal cord, we found less than a 5% overlap between these markers (data not shown). SCI animals also exhibited immunoreactivity for some complement proteins, particularly C1q and FB, on structures that appeared to be axon fibers in the dorsal, lateral, and ventral funiculi. No labeling for terminal complement (C5b-9) was observed on these structures. Triple labeling for C1q or FB, the cytoskeletal marker SMI 31, and DAPI showed these immunoreactive profiles to be axon fibers (Fig. 4). Note that not all SMI 31–positive axons were complement-immunoreactive, supporting the specificity of labeling.

Section Area and Quantification of Cellular Labeling Previous studies have shown that the extent of NYU Impactor contusion-induced SCI lesions, as measured by length and/or volume, changes dramatically over time in and near the lesion epicenter (Rabchevsky et al., 2002). However, this effect decreases with distance, in accordance with the restriction of the lesion to a central area surrounding the epicenter. The regions selected for quantification of cellular labeling in the present study were first determined to be cytoarchitecturally normal based on LFB/H&E staining, that is, outside the primary lesion area. However, quantification and statistical analysis using 2-way ANOVA revealed significant interactions between section area and spinal cord level and/or injury severity (Fig. 5). Gray matter section area  spinal cord

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FIG. 1. Contusion-induced SCI results in complement activation. C1q (A,B), C4 (C,D), FB (E,F), and C5b-9 (G,H). Note the lack of immunoreactivity in laminectomy controls (A,C,E,G). in contrast with the intense staining observed in SCI animals (B,D,F,H). SCI animals shown were subjected to mild contusion injury using the NYU Impactor (12.5 mm weight drop). All sections shown are from animals sacrificed 1 day post-injury. Bar  500 m.

level df  2, F  87.756, p  0.0001; gray matter section area  injury severity df  5, F  14.817, p  0.0001; gray matter section area  spinal cord level  injury severity df  10, F  6.121, p  0.0001. White matter section area  spinal cord level df  2, F  6.043, p  0.0043; white matter section area  injury severity df  5, F  9.523, p  0.0001; white matter section area  spinal cord level  injury severity df  10, F  1.104, p  0.3673. Total cross-section area  spinal cord level df  2, F  24.031, p  0.0001; total

cross-section area  injury severity df  5, F  12.472, p  0.0001; total cross-section area  spinal cord level  injury severity df  10, F  2.083, p  0.0420. Based on these results, expressing cellular labeling per mm2 could be predicted to underestimate the number of labeled cells 1 day post-SCI and overestimate the number of labeled cells 42 days post-SCI, in accordance with changes in tissue area at these timepoints. Consequently, instead of box or grid-based sampling of small areas within a section

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FIG. 2. C1q, C4, FB, and C5b-9 immunolabel neurons and glia in the contusion-injured rat spinal cord. C1q (A,B), C4 (C,D), FB (E,F), and C5b-9 (G,H). Arrows indicate cellular labeling in gray matter (A,C,E,G). and white matter (B,D,F,H). Note that complement protein labeling in gray matter appears selective to profiles with neuronal morphology, labeling of gray matter astrocytes is not apparent. Arrowheads in B and F indicate punctate staining in white matter in the case of C1q and FB. All images are from mild SCI animals (12.5 mm weight drop), 1 day post-injury. Bar  50 m.

FIG. 3. APC/CC-1-positive oligodendrocytes are complement immunoreactive after SCI. (A,C) Triple labeling for C1q (A), the oligodendrocyte/O2-A precursor marker APC/CC-1 (B), and the nuclear stain DAPI (C). D–F Triple labeling for FB (D), CC-1, (E) and the nuclear stain DAPI (F). Similar observations were found for the terminal pathway marker C5b-9 (not shown). Arrows indicate triple-labeled profiles. Arrowheads indicate examples of single-labeled profiles. The morphology of complement protein-positive, CC-1–positive cells observed suggests that these cells are predominantly oligodendrocytes. Bar  50 m. FIG. 4. SMI 31–positive axon profiles are complement immunoreactive after SCI. Triple labeling for C1q (A), the neuronal cytoskeletal marker SMI 31 (B), and the nuclear stain DAPI (C). Triple labeling for FB (D), SMI 31 (E), and the nuclear stain DAPI (F). Arrowheads indicate examples of complement-positive profiles for comparison with corresponding SMI 31 staining; note that not all SMI 31–positive axon profiles are immunoreactive for complement. Arrows indicate nuclei that are complementpositive and SMI 31–negative. Bar  50 m.

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FIG. 3.

FIG. 4.

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Quantification of Cellular Labeling: Timecourse and Injury Severity

FIG. 5. Gray matter, white matter, and total section area change with time and injury severity outside the direct area of spinal cord injury. Gray matter area (A), white matter area, (B), and total section area (C) were obtained from hemisections corresponding to those analyzed for complement protein immunostaining. Clear differences in these measures were apparent across time and injury severity, as determined by 2-way ANOVA and Fisher’s PLSD. N  4 animals for each injury condition and post injury timepoint, as described under Methods. Data shown are mean
SEM. *p  0.05, **p  0.005, ***p  0.0001 for spinal cord level, injury severity, and spinal cord level  injury severity (interaction). as indicated.

to determine the relative numbers of labeled cells per animal, all of the labeled cells in each hemisection were counted as described under Methods, and the average number of complement-labeled cells in each animal was expressed per hemisection rather than per unit area (mm2).

Consistent with qualitative observation of stained sections and the lack of complement immunoreactivity in uninjured spinal cord, analysis of gray matter neuronal and white matter oligodendrocyte immunoreactivity in mild (12.5 mm, black bars) and severe (50 mm, white bars) contusion-injured rats demonstrated complement antigens (cells/hemisection) were greatly increased following SCI in comparison with laminectomy controls at most post-injury timepoints (Fig. 6). Changes in complement labeling in injured versus laminectomy control animals ranged from a 3–12-fold change in the number of labeled neuronal or oligodendroglial cells per hemisection. Statistical analysis using 1-way ANOVA (df  6) and post-hoc Fisher PLSDs revealed significant differences in the number of gray matter neurons for C1q (F  6.480, p  0.0001), C4 (F  5.369, p  0.0001), FB (F  6.566, p  0.0001), and C5b-9 (F  7.451, p  0.0001), and white matter oligodendroglia for C1q (F  3.842, p  0.0021), C4 (F  4.904, p  0.0003), FB (F  5.945, p  0.0001), and C5b-9 (F  7.284, p  0.0001). The number of complement-labeled cells varied with the specific complement antigen assessed, however, overall similarities in the timecourse and pattern of labeling were apparent (Fig. 6). First, C1q and FB immunostaining revealed the greatest number of labeled neurons in the gray matter, followed by C4 and C5b-9. In contrast, the numbers of labeled oligodendroglia in the white matter were comparable for C1q, FB, C4, and C5b-9. Second, complement immunoreactivity was frequently similar between mild and severe injuries; differences between injury severities rarely reached statistical significance at the timepoints investigated (indicated by #, Fig. 6). Third, animals subjected to severe contusion injury exhibited a relatively stable profile of complement immunoreactivity across the 1 day, 7 days, and 42 days timecourse (Fig. 6, white bars). In contrast, animals with mild contusion injuries tended to exhibit a biphasic distribution of complement immunoreactivity (Fig. 6, black bars), in which the greatest complement immunoreactivity was apparent at 1 and 42 days post-SCI. This pattern was particularly evident for MAC/C5b-9, and was consistent with morphological observations of the sections. Additionally, although rostral and caudal regions of the spinal cord (C2, R2, R4 above) are not presented in separate analyses because the number of complement-immunoreactive cells could be influenced by differential changes in section area, ANOVA analysis of rostral-caudal level  complement immunoreactive cells  injury severity did not

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FIG. 6. Effect of post-injury time and severity of injury on complement-positive cell number. Tissue sections were stained for C1q (A,B), C4 (C,D), FB (E,F), and C5b-9 (G,H). Gray matter neurons (A,C,E,G) and white matter glia (B,D,F,H) were counted in spinal cord hemisections in animals with mild or severe SCI at 1, 7, and 42 days post-injury (dpi). N  4 animals for each injury condition and post-injury timepoint. SCI animals were compared with laminectomy controls sacrificed 1 day post-surgery, N  4 animals as above. Bars indicate quantification for mild injury (black, 12.5 mm weight drop), severe injury (white, 50 mm weight drop) and laminectomy control (gray) groups. Data shown are mean
SEM. Laminectomy control means and SEMs  0 as no labeling was observed in these animals. *p  0.05 in comparison with laminectomy controls, #p  0.05 in comparison with mild SCI at the same timepoint. Data analyzed using ANOVA and post-hoc Fisher’s PLSD.

yield statistical significance (C1q gray matter p  0.8627, C1q white matter p  0.9989, C4 gray matter p  0.9423, C4 white matter p  7, FB gray matter p  0.9575, FB white matter p  0.9904, C5b-9 gray matter p  0.9934, C5b-9 white matter p  0.9999), supporting the observation that complement immunoreactivity is broadly distributed after SCI.

DISCUSSION Complement Synthesis and Deposition This study investigated activation of the complement cascade as a component of the immunologic response to acute SCI. Complement immunoreactivity was found in association with oligodendrocytes, neurons, and axons in

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ANDERSON ET AL. injured, but not laminectomy control, spinal cord. The prolonged timecourse of complement immunoreactivity, extending for at least 6 weeks after the acute injury, was particularly surprising and suggests an extended activation of this inflammatory mechanism after SCI. Peripheral nerve transection, but not dorsal root transection, has previously been shown to increase immunoreactivity for C1q and other complement components in the ipsilateral gracile nucleus and dorsal horn (Liu et al., 1995, 1998; Törnqvist et al., 1996). In contrast to our findings, changes in complement expression in this paradigm were predominantly found in microglia, neither neuronal nor axonal complement immunoreactivity was induced. Based on these observations, Liu et al. have suggested that anterograde/Wallerian degeneration in the spinal cord, in the absence of BBB opening, does not activate complement. In this regard, there are three principal potential sources for complement in the injured spinal cord: cellular synthesis by CNS resident cells, cellular synthesis by invading immune cells, and BBB/BSB opening. CNS astrocytes, microglia/macrophages, oligodendrocytes, and neurons are capable of synthesizing many, perhaps all, of the components required for a complete and functional complement system (Barnum, 1999). This source of complement synthesis may thus be an important factor in the response to injury and/or resolution of infection in the CNS (Gasque et al., 1995). In this regard, brain contusion, excitotoxic or neurotoxic insult, and perforant path transection have been reported to increase complement expression in CNS glia and neurons (Bellander et al., 1996; Johnson et al., 1996; Pasinetti et al., 1992; Rozovsky et al., 1994; Singhrao et al., 1999; Van Beek et al., 2000). Thus, spinal cord resident cells are capable of producing all of the components of the classical, alternative and terminal complement pathways observed in the present study. Additional experiments investigating complement mRNA synthesis via in situ hybridization will be necessary to establish whether resident CNS cells contribute to observation of complement in the injured spinal cord. Similarly, the rapid infiltration of inflammatory cells and induction of cytokine production in the cord following injury is consistent with a potential role for infiltrating inflammatory cell synthesis, secretion, and deposition of complement proteins (Carlson et al., 1998; Dusart and Schwab, 1994; Popovich et al., 1997). However, while infiltrating microglia/macrophages exhibit a rostral-caudal gradient in response timecourse, peaking within 3–7 days at the lesion epicenter and within 1–4 weeks rostral and caudal to the injury, the complement response was maximal within 1 day at all levels of the spinal cord examined. Additionally, macrophage/mi-

croglial cells appear predominantly restricted to white matter in degenerating fiber tracts at later timepoints (1–2 weeks) (Popovich et al., 1997), which is not consistent with the distribution of complement proteins throughout the gray and white matter observed in the present study. This lack of concordance suggests that an alternative source of complement, e.g., resident cell synthesis or BBB/BSB opening, must also play a role. Complement levels in plasma/serum are generally several hundredfold higher than in CSF, reflecting the “immune-privileged” status of the CNS. Contact between these two physiological compartments, plasma/serum and the CNS/CSF, can trigger complement activation and MAC assembly under both in vitro and in vivo conditions. Consequently, such intercompartmental contact may affect the regulatory balance of the complement system, promoting inflammatory cell recruitment and exacerbating injury in turn (Lindsberg et al., 1996). Previous studies using macromolecular (e.g., HRP) tracers or immunoreactivity for anti-rat IgGs have suggested that BSB permeability is increased for a period of 1–2 weeks following acute SCI but resolved thereafter (Dusart and Schwab, 1994; Noble and Wrathall, 1989). In contrast, small molecule tracers have suggested that BSB permeability is increased in the dorsal columns, corticospinal tracts, and gray matter lamina within 3 days, and persists for at least 4 weeks, after contusion-induced SCI (Popovich et al., 1996). Altered BSB permeability was observed to progressively spread along the rostral-caudal spinal cord axis with increasing post-injury survival time, reaching distances of up to 30 mm from the lesion epicenter. However, BSB permeability changes were not observed in the ventral or lateral white matter until 14–28 days post-injury. This finding is in contrast to the localization of gray matter neuronal and white matter oligodendroglial complement immunoreactivity at all levels of the spinal cord within 1 day of injury in the present study, again suggesting that BBB/BSB changes are unlikely to be the sole source of complement in the injured spinal cord. A further possibility is that localized changes in BSB saturable transport mechanisms for inflammatory cytokines such as interferon-, interferon-, TNF- IL1, and IL-1
could play a more specific role in enhancing cellular synthesis of complement proteins, even in areas of the cord that do not exhibit gross BSB breakdown. Several of these transport mechanisms have been shown to be altered in a regionally specific manner following acute SCI or systemic cytokine administration (Pan et al., 1997a,b; Saija et al., 1995).

Timecourse of Complement Activation The prolonged timecourse of complement immunoreactivity, extending for at least 6 weeks after the acute in-

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SCI-INDUCED COMPLEMENT ACTIVATION jury, is a particularly surprising finding that suggests an extended activation of this inflammatory mechanism after SCI. Interestingly, cytokine mRNA expression after SCI returns to control levels within 1 day (including IL1, IL-1
, TNF-, M-CSF, and MIP-1) to 2 weeks (TGF
1) (Bartholdi and Schwab, 1997; Streit et al., 1998), and changes in cytokine mRNA expression are restricted to the lesion site (Bartholdi and Schwab, 1997). However, these studies are unable to address the entry of cytokine proteins across the BBB as a part of the systemic immune response, thus, the true timecourse and distribution of bioavailable cytokines after SCI is unknown. The kinetics of the cellular/cytokine inflammatory response, the biphasic pattern of complement immunoreactivity observed, and the potential for multiple sources of complement proteins to contribute to these observations, suggest a multiphasic inflammatory response to SCI. For example, initial membrane deposition of circulating complement proteins from plasma/serum could be followed by the synthesis of complement proteins in resident oligodendrocytes and/or neurons. The attenuation of a biphasic pattern of complement immunoreactivity following a more severe contusion could reflect either a more consistent activation of infiltrating cells, a more rapid induction of complement synthesis in resident cells, or an accelerated/sustained change in BSB permeability.

Role of Complement Activation in the Cellular Inflammatory Response This study demonstrates the activation of both the classical (C1q, C4) and alternative (FB) complement pathways after acute SCI. These enzymatic cascades form complexes (e.g., MAC and the C3 and C5 convertases) and proteolytic cleavage products (e.g., the C3a, C4a and C5a anaphylatoxins), which mediate a variety of inflammatory functions relevant to SCI. These include mast cell and basophil degranulation and pro-inflammatory molecule release, phagocyte and neutrophil chemotaxis, upregulation of neutrophil adhesion molecules for neutrophil recruitment, activation and stimulation of oxidative burst in neutrophils, opsonization of cells and debris for phagocytic clearance, and changes in vascular permeability (Barnum, 1999; Makrides, 1998; Morgan, 1995). The complement cascade also regulates, and is regulated by, other inflammation pathways. Pro-inflammatory cytokines such as interferon-, tumor necrosis factor–, interleukin-1
, interleukin-6, and interleukin13 can upregulate the expression of complement proteins, including C1r, C3, C4, C1-inhibitor, or FB. Moreover, complement components such as C1q, C3a, C5a, and MAC can upregulate the expression or enhance the stimulation of multiple cytokines and chemokines, including interleukin-6, interleukin-8, RANTES, and MCP-1. Im-

portantly, recent studies in reperfused myocardium suggest that monocyte chemotaxis is driven sequentially, first by activated C5a within the first hour, followed by the cytokine TGF-
at 1–3 h and the chemokine MCP-1 after 3 h (Birdsall et al., 1997). Rapid opening of the BSB after SCI is likely to provide immediate access of circulating complement to the site of injury, suggesting that a similar mechanism could be operative.

Axonal Complement Deposition Complement activation in association with axons may be particularly interesting in the context of SCI. Axonal C1q and FB in this study could in theory reflect either myelin or cytoskeletal deposition. The association of complement with neuronal fibers in both the CNS and PNS has been observed in association with a variety of neurological diseases, but has generally been thought to be in relationship to myelin (Ferrari et al., 1998; Itagaki et al., 1994; Lindsberg et al., 1996; Singhrao et al., 1999; Storch et al., 1998; Webster et al., 1997). It is well established that myelin phagocytosis by microglia and macrophages is stimulated by complement-mediated opsonization, additionally, binding inhibition studies show that C3 receptors can replace antibody-antigen complexing to stimulate phagocytosis (Brück and Friede, 1990; Dejong and Smith, 1997; Mosley and Cuzner, 1996; Van Der Laan et al., 1996). Furthermore, direct binding of C3 by myelin-associated proteins, as has been demonstrated for cyclic nucleotide phosphodiesterase (CNP), may be an important event for demyelination in multiple sclerosis or after injury (Walsh and Murray, 1998). Myelin has been known to activate the classical complement pathway in an antibody-independent manner for some time (Vanguri et al., 1982), further, myelin oligodendrocyte glycoprotein (MOG) has been shown to bind C1q with dose-dependent and saturable kinetics (Johns and Bernard, 1997), suggesting one mechanism for direct complement activation. In addition, terminal complement activation and MAC assembly are associated with morphological disruption of the myelin sheath and macrophage-mediated demyelination in vitro (Brück et al., 1995), and recent data has shown a connection between membrane attack complex formation and demyelination in EAE (Mead et al., 2002). Taken together, these studies suggest that antibody-dependent or independent activation of complement pathways play a key role in the clearance of myelin debris, and perhaps in active demyelination, in disease and/or following injury.

Membrane Attack Complex Deposition In addition to labeling for members of the classical and alternative complement pathways, C5b-9 labeling in con-

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ANDERSON ET AL. tused spinal cord strongly suggests activation of the terminal pathway. C5b-9 antibody is raised against a purified human C9, and has been shown to bind a neoantigen expressed on C5b-9 complex (MAC) and poly-C9, both of which are indicative of membrane attack complex formation. While early phase complement proteins such as C1q and C3 serve to opsonize cells and debris for phagocytosis, MAC assembly (C5b-9) can cause lytic cell death. In this context, neurons and oligodendrocytes are both vulnerable to complement-mediated cell death (Agoropoulou et al., 1996; Scolding et al., 1989; Shen et al., 1995; Wren and Nobel, 1989), suggesting that complement activation can exacerbate damage in the injured CNS by contributing to demyelination and neurodegeneration. Additionally, the C5a anaphylatoxin can also induce apoptosis via a receptor-mediated signaling pathway (Farkas et al., 1998a,b). However, injury-induced MAC deposition in the CNS could exhibit a prolonged timecourse that is not necessarily indicative of cell death per se. Many cells, including oligodendrocytes, tolerate a sub-lethal level of MAC deposition (Morgan, 1989), and membrane-bound inhibitors (e.g., CD59) as well as soluble factors can protect oligodendrocytes and neurons from MAC-driven lysis (Agoropoulou et al., 1996, 1998; Shen et al., 1997). Additionally, many cells including oligodendrocytes and possibly neurons, can clear and degrade MACs via endocytosis (Itagaki et al., 1994; Morgan, 1989). Although the role of sublethal MAC remains unclear, MAC deposition has been shown to alter calcium homeostasis and cellular signaling pathways (Morgan, 1989). Interestingly, evidence suggests that oligodendrocyte injury resulting from a single sub-lytic complement-mediated attack may be reversible, while repeated or prolonged attacks may ultimately result in cell death (Scolding et al., 1989). Thus, although the terminal complement pathway seems likely to contribute to neuronal and oligodendrocyte degeneration, sublethal MAC deposition on neurons/oligodendrocytes may also play a role in the cellular response to injury.

Role of Complement in CNS Degeneration and Regeneration In accordance with the predicted degenerative consequences of complement activation on local cells, complement inhibition is beneficial in several models of CNS injury, for example, traumatic brain injury (Hicks et al., 2002; Rancan et al., 2003), experimental allergic encephalomyelitis (Davoust et al., 1999; Jung et al., 1995; Piddlesden et al., 1994), cerebral ischemia (Huang et al., 1999; Vasthare et al., 1998), and experimental autoimmune myasthenia gravis (Piddlesden et al., 1996). In contrast, complement activation may also be beneficial to regeneration and functional recovery in some instances. For

example, complement-induced demyelination promotes regeneration after lateral hemisection (Dyer et al., 1998), and knife cut axonal injury (Keirstead et al., 1998), in the adult rat. Similarly, complement depletion inhibits regeneration after peripheral nerve injury (Dailey et al., 1998). Such a positive role for complement activation in regeneration may reflect the necessity of clearing cellular debris (via opsonization and mediation/stimulation of phagocytosis) and molecules inhibitory to regeneration from the area of injury in order to create a permissive environment for axonal outgrowth. In this light, Liu et al. (1998) have previously suggested that the capacity for regeneration following peripheral nerve injury may be related to complement-driven myelin clearance. Additionally, sublytic C5b-9 is likely to contribute to both demyelination and recovery, by downregulating myelin basic protein expression and inducing oligodendrocyte cell cycle re-entry and proliferation (Rus et al., 1996; Shirazi et al., 1993), and correspondingly downregulating P0 and inducing schwann cell proliferation (Dashiell and Koski, 1999; Dashiell et al., 2000). Interestingly, sublytic C5b-9 also inhibits apoptosis in oligodendrocyte precursors and schwann cells (Dashiell and Koski, 1999; Soane et al., 1999), and under some conditions, may protect oligodendrocytes from death via intracellular regulation of Bad, enhanced Bcl-2 synthesis, and caspase inhibition (Soane et al., 1999, 2001). These findings illustrate the complexity of the complement response, the mediation of numerous cellular and systemic effects by the complement cascade and its products, and the need for better understanding of this aspect of the inflammatory cascade.

CONCLUSION In the present study, we have described the activation of complement after contusion injury to the rat spinal cord. However, the cellular regulation of complement proteins remains to be defined and the role of complement components and pathways in degenerative and regenerative events remains to be established. In this light, the complex regulation of complement in vivo, and multipathway/multiphasic activation of complement observed after SCI in the present study may suggest that selective complement inhibition could be a viable therapeutic target for the suppression of the negative effects of inflammation after SCI.

ACKNOWLEDGMENTS This work was supported by the Christopher Reeve Paralysis Foundation Consortium on Spinal Cord Injury

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SCI-INDUCED COMPLEMENT ACTIVATION (to C.W.C.), the Paralysis Project of America (PPA28589, to A.J.A.), and the National Institutes of Health (R01 043428-01A1, to A.J.A.).

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