Title: Complement Plays an Important Role in Spinal Cord Injury and Represents a Therapeutic Target for Improving Recovery following Trauma
Abstract: Initiation of an inflammatory cascade following traumatic spinal cord injury (SCI) is thought to cause secondary injury and to adversely impact functional recovery, although the mechanisms involved are not well defined. We report on the dynamics of complement activation and deposition in the mouse spinal cord following traumatic injury, the role of complement in the development of SCI, and the characterization of a novel targeted complement inhibitor. Following traumatic injury, mice deficient in C3 had a significantly improved locomotor score when compared with wild-type controls, and analysis of their spinal cords revealed significantly more tissue sparing, with significantly less necrosis, demyelination, and neutrophil infiltration. Wild-type mice were also treated with CR2-Crry, a novel inhibitor of complement activation that targets to sites of C3 deposition. A single intravenous injection of CR2-Crry 1 hour after traumatic injury improved functional outcome and pathology to an extent similar to that seen in C3-deficient animals. CR2-Crry specifically targeted to the injured spinal cord in a distribution pattern corresponding to that seen for deposited C3. As immunosuppression is undesirable in patients following SCI, targeted CR2-Crry may provide appropriate bioavailability to treat SCI at a dose that does not significantly affect systemic levels of serum complement activity. Initiation of an inflammatory cascade following traumatic spinal cord injury (SCI) is thought to cause secondary injury and to adversely impact functional recovery, although the mechanisms involved are not well defined. We report on the dynamics of complement activation and deposition in the mouse spinal cord following traumatic injury, the role of complement in the development of SCI, and the characterization of a novel targeted complement inhibitor. Following traumatic injury, mice deficient in C3 had a significantly improved locomotor score when compared with wild-type controls, and analysis of their spinal cords revealed significantly more tissue sparing, with significantly less necrosis, demyelination, and neutrophil infiltration. Wild-type mice were also treated with CR2-Crry, a novel inhibitor of complement activation that targets to sites of C3 deposition. A single intravenous injection of CR2-Crry 1 hour after traumatic injury improved functional outcome and pathology to an extent similar to that seen in C3-deficient animals. CR2-Crry specifically targeted to the injured spinal cord in a distribution pattern corresponding to that seen for deposited C3. As immunosuppression is undesirable in patients following SCI, targeted CR2-Crry may provide appropriate bioavailability to treat SCI at a dose that does not significantly affect systemic levels of serum complement activity. Spinal cord injury (SCI) is characterized by an initial traumatic injury phase, followed closely by secondary events that result in edema, ischemia, excitotoxicity, and inflammation.1Kwon BK Tetzlaff W Grauer JN Beiner J Vaccaro AR Pathophysiology and pharmacologic treatment of acute spinal cord injury.Spine J. 2004; 4: 451-464Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar The mechanisms of secondary injury are not well defined, but it is clear that inflammatory processes play a significant role in functional recovery.1Kwon BK Tetzlaff W Grauer JN Beiner J Vaccaro AR Pathophysiology and pharmacologic treatment of acute spinal cord injury.Spine J. 2004; 4: 451-464Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 2Taoka Y Okajima K Uchiba M Murakami K Kushimoto S Johno M Naruo M Okabe H Takatsuki K Role of neutrophils in spinal cord injury in the rat.Neuroscience. 1997; 79: 1177-1182Crossref PubMed Scopus (293) Google Scholar While the initial traumatic injury is difficult to guard against, the subsequent inflammatory cascade represents a therapeutic target for SCI. The only clinical therapy accepted currently for acute SCI is methylprednisolone, a therapy that has yielded disappointing results, with the data from clinical trails being contradictory and inconclusive.3Fiore C Inman DM Hirose S Noble LJ Igarashi T Compagnone NA Treatment with the neurosteroid dehydroepiandrosterone promotes recovery of motor behavior after moderate contusive spinal cord injury in the mouse.J Neurosci Res. 2004; 75: 391-400Crossref PubMed Scopus (60) Google Scholar, 4Tsai EC Tator CH Neuroprotection and regeneration strategies for spinal cord repair.Curr Pharm Des. 2005; 11: 1211-1222Crossref PubMed Scopus (36) Google Scholar, 5Hall ED Springer JE Neuroprotection and acute spinal cord injury: a reappraisal.NeuroRx. 2004; 1: 80-100Crossref PubMed Scopus (336) Google ScholarComplement activation, by any one of three pathways (classical, alternative, or lectin), converges at the cleavage of C3 by C3 convertases and then proceeds in a common pathway to form the terminal proinflammatory and cytolytic membrane attack complex (MAC). Other biologically active by-products of this process are C3 opsonins that bind receptors on immune effector cells and the soluble chemotactic and proinflammatory mediators C3a and C5a. Little is known about the role of complement in inflammation and secondary injury following traumatic SCI. Recent studies by Anderson et al6Anderson AJ Najbauer J Huang W Young W Robert S Upregulation of complement inhibitors in association with vulnerable cells following contusion-induced spinal cord injury.J Neurotrauma. 2005; 22: 382-397Crossref PubMed Scopus (21) Google Scholar demonstrated that complement proteins are deposited at sites of SCI on neurons and oligodendrocytes for a sustained period following injury in rats. In a subsequent study, it was shown that the complement inhibitory proteins factor H and clusterin were present at increased levels among neurons and oligodendrocytes after SCI in rats, and it was suggested that these complement inhibitors function to limit the inflammatory reaction in the injured spinal cord.7Anderson AJ Robert S Huang W Young W Cotman CW Activation of complement pathways after contusion-induced spinal cord injury.J Neurotrauma. 2004; 21: 1831-1846Crossref PubMed Scopus (90) Google Scholar Other data implicating complement in the pathogenesis of SCI come from therapeutic studies, also performed in rats, showing that intraparenchymal infusion of vaccinia virus complement control protein (VCP)8Reynolds DN Smith SA Zhang YP Lahiri DK Morassutti DJ Shields CB Kotwal GJ Vaccinia virus complement control protein modulates inflammation following spinal cord injury.Ann NY Acad Sci. 2003; 1010: 534-539Crossref PubMed Scopus (15) Google Scholar, 9Reynolds DN Smith SA Zhang YP Mengsheng Q Lahiri DK Morassutti DJ Shields CB Kotwal GJ Vaccinia virus complement control protein reduces inflammation and improves spinal cord integrity following spinal cord injury.Ann NY Acad Sci. 2004; 1035: 165-178Crossref PubMed Scopus (39) Google Scholar improves cord integrity and motor function.The objectives of the current study were twofold. First, to investigate the dynamics of complement activation and its role in the development of SCI in mice, a more versatile model than the rat for studying disease processes. To address this we used a contusion-induced model of SCI in normal mice and in mice deficient in the third component of complement (C3). The second objective was to investigate the neuroprotective effect of a novel targeted complement inhibitor that is administered intravenously. For the therapeutic studies we used mouse CR2-Crry, a complement inhibitory fusion protein that functions at the C3 level. The complement inhibitor, Crry, is targeted specifically to sites of complement activation by means of the complement receptor 2 (CR2)-targeting moiety.10Atkinson C Song H Lu B Qiao F Burns TA Holers VM Tsokos GC Tomlinson S Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.J Clin Invest. 2005; 115: 2444-2453Crossref PubMed Scopus (144) Google Scholar CR2 is a member of the C3-binding protein family, and its natural ligands are cleavage fragments of C3 that become deposited at sites of complement activation. Targeted complement inhibition has been shown to provide significant benefits over systemic (untargeted) inhibition in terms of efficacy and host susceptibility to infection.11Kuhn PL Wrathall JR A mouse model of graded contusive spinal cord injury.J Neurotrauma. 1998; 15: 125-140Crossref PubMed Scopus (112) Google Scholar The use of mouse Crry is appropriate when studying the effects of complement inhibition in mice, because complement inhibitors display different degrees of species selectivity. Crry is a structural and direct functional analog of human complement receptor 1 (CR1), and data obtained with Crry in rodents will likely translate in functional terms to the use of CR1 in humans.Materials and MethodsCR2-Crry Complement InhibitorThe fusion protein CR2-Crry was produced and purified as described previously.10Atkinson C Song H Lu B Qiao F Burns TA Holers VM Tsokos GC Tomlinson S Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.J Clin Invest. 2005; 115: 2444-2453Crossref PubMed Scopus (144) Google Scholar In brief, a cDNA construct of the recombinant fusion protein was prepared by joining the mouse CR2 sequence encoding the four N-terminal short consensus repeat (SCR) units (residues 1–257 of mature protein, National Center for Biotechnology Information GenBank, accession number M35684) to sequences encoding extracellular regions of mouse Crry. The Crry sequence used encoded residues 1–319 of the mature protein (National Center for Biotechnology Information GenBank, accession number NM013499). To join CR2 to Crry, linking sequences encoding (GGGGS)2 were used. The recombinant protein was expressed in NSO cells and purified by anti-Crry affinity chromatography as described.10Atkinson C Song H Lu B Qiao F Burns TA Holers VM Tsokos GC Tomlinson S Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.J Clin Invest. 2005; 115: 2444-2453Crossref PubMed Scopus (144) Google Scholar CR2-Crry has a circulatory half-life in C57BL/6 mice of ∼8 hours.10Atkinson C Song H Lu B Qiao F Burns TA Holers VM Tsokos GC Tomlinson S Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection.J Clin Invest. 2005; 115: 2444-2453Crossref PubMed Scopus (144) Google ScholarAnimals and SCI ModelFemale wild-type C57Bl/6 and C57Bl/6 C3-deficient mice (Jackson Laboratories, Bar Harbor, ME), weighing 16 to 20 g and between 6 and 8 weeks old were used in this study. C57Bl/6 wild type were randomized into sham (laminectomy, no SCI damage), vehicle control (phosphate-buffered saline [PBS]), and CR2-Crry treatment groups. Another group using C3-deficient animals was also included. Each animal was anesthetized with 10 mg/kg ketamine and 6 mg/kg xylazine by intraperitoneally injection. Animals were breathing spontaneously, and body temperature was maintained using a heat mat for the duration of the experiment. A laminectomy at T12 was performed, and the dura was exposed using aseptic technique. A weight-drop contusion injury was performed (5 g, 3 cm) using the procedure previously described.11Kuhn PL Wrathall JR A mouse model of graded contusive spinal cord injury.J Neurotrauma. 1998; 15: 125-140Crossref PubMed Scopus (112) Google Scholar An initial study assessed the presence, time course, and activation of complement after SCI in C57Bl/6 mice. Animals (n = 3) underwent SCI and were sacrificed at 1-, 2-, 4-, 24-, and 48-hours after injury, and complement activation was assessed by the presence of deposited C3. On completion of these initial experiments four groups of animals (sham, vehicle control, CR2-Crry-treated, and C3-deficient mice) were subjected to SCI. Following injury, animals were allowed to recovery for 1 hour at which point animals randomized to CR2-Crry treatment were administered a single dose of 0.25 mg of CR2-Crry by tail vein injection. All other animals received intravenous injections of phosphate buffered saline. Following injury animals were monitored for locomotor recovery and animals in different groups sacrificed at 24 hours, 72 hours, 7 days, and 21 days. All procedures were approved by the Medical University of South Carolina's committee for animal research, in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.Locomotor RecoveryLocomotor recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) rating scale developed for rats,12Basso DM Beattie MS Bresnahan JC Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection.Exp Neurol. 1996; 139: 244-256Crossref PubMed Scopus (1249) Google Scholar but later adapted by others for mice.13Noble LJ Donovan F Igarashi T Goussev S Werb Z Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events.J Neurosci. 2002; 22: 7526-7535PubMed Google Scholar, 14Mikami Y Toda M Watanabe M Nakamura M Toyama Y Kawakami Y A simple and reliable behavioral analysis of locomotor function after spinal cord injury in mice. Technical note.J Neurosurg. 2002; 97: 142-147PubMed Google Scholar, 15Wells JE Rice TK Nuttall RK Edwards DR Zekki H Rivest S Yong VW An adverse role for matrix metalloproteinase 12 after spinal cord injury in mice.J Neurosci. 2003; 23: 10107-10115Crossref PubMed Google Scholar, 16Wells JE Hurlbert RJ Fehlings MG Yong VW Neuroprotection by minocycline facilitates significant recovery from spinal cord injury in mice.Brain. 2003; 126: 1628-1637Crossref PubMed Scopus (341) Google Scholar The BBB locomotor rating scale is an open-field 21 point evaluation and is rated according to categories describing the quality of joint movements, the trunk, abdomen, and paw placement, stepping, trunk stability, and tail position. Animals were assessed preoperatively, on the day of surgery, and then daily postoperatively by an observer blinded to animal treatment. Changes in BBB score during spinal cord injury between vehicle control, C3-deficient, CR2-Crry-treated, and control animals were determined by analysis of variance with repeated measures using Scheff's test for posthoc comparisons. A P value of less than 0.05 was considered statistically significant. All data were subjected to statistical analysis using Statview Analysis Software (version 5; SAS Institute Inc., Cary, NC).HistologyAnimals were transcardially perfused with 4% paraformaldehyde at 24 hours, 72 hours, 7 days, and 21 days after injury, and spinal cords removed. Spinal cords were either frozen for cryosectioning or processed in paraffin using standard techniques.17Carlson SL Parrish ME Springer JE Doty K Dossett L Acute inflammatory response in spinal cord following impact injury.Exp Neurol. 1998; 151: 77-88Crossref PubMed Scopus (432) Google Scholar Morphometric analyses to determine the degree of tissue damage following injury were conducted using transverse sections of spinal cord stained with a standard hematoxylin and eosin (H&E) stain. The cross-sectioned area of spinal cord was measured at 100-μm increments extending 2 mm either side of the injury epicenter, and averaged for animals in each group and time point as previously described.18Gonzalez R Glaser J Liu MT Lane TE Keirstead HS Reducing inflammation decreases secondary degeneration and functional deficit after spinal cord injury.Exp Neurol. 2003; 184: 456-463Crossref PubMed Scopus (141) Google Scholar The presence of myelin damage was assessed by staining transverse sections of the spinal cord at the epicenter of injury with the Luxol fast blue stain.17Carlson SL Parrish ME Springer JE Doty K Dossett L Acute inflammatory response in spinal cord following impact injury.Exp Neurol. 1998; 151: 77-88Crossref PubMed Scopus (432) Google Scholar All assessments were performed by a pathologist in a blinded fashion.Immunofluorescence Staining for Complement Component C3, CR2-Crry, and NeutrophilsCryosections were fixed in cold acetone for 5 minutes and then washed in running water followed by PBS. Sections were then incubated for 1 hour at room temperature with anti-mouse C3 fluorescein isothiocyanate (FITC), anti-mouse neutrophil antibody clone number (GR) 1 FITC (BD Pharmingen, San Diego, CA), or anti-mouse CR2 (Santa Cruz Biotechnology, Santa Cruz, CA) and then washed in three changes of PBS. C3- and GR1-stained sections were coverslipped with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) and analyzed using a Zeiss LSM5 Confocal microscope (Carl Zeiss, Oberkochen, Germany). CR2-stained sections were then incubated with rabbit anti-goat biotinylated antibody (Vecta Laboratories, Burlingame, CA) and then washed before being incubated with a streptavidin FITC (Vecta Laboratories). Sections were coverslipped and analyzed as described above. The presence of complement component C3 was assessed at the injury site and caudal and rostral to injury, in transverse sections of spinal cord harvested at all time points. Neutrophils were quantified at the epicenter, defined as the section exhibiting maximal tissue damage. All counts were performed with the observer blinded to the experimental group. The total number of neutrophils/mm2 was determined at the epicenter. No attempt was made to differentiate between neutrophil infiltration into white or gray matter because of the early dissolution of tissue at the injury site at early time points. Neutrophils were assessed in spinal cords from all treatment groups at 24 hours, 72 hours, and 7 days after injury. CR2 staining was used to identify the presence of CR2-Crry protein at the site of injury in the treatment group and control animals 12 hours after injury.125I Radiolabeling and BiodistributionRadiolabeling was conducted using 125I (Amersham Biosciences, Pittsburgh, PA) by the Iodo-Gen method (Pierce Chemical, Rockford, IL), with 5 mCi used to label 100 μg of CR2-Crry protein. Free iodine was removed from the mixture after labeling by anion-exchange resin. Iodine incorporation was in the 50 to 80% range. Radiolabeled protein was injected intravenously for 1 hour after SCI to C57Bl/6 mice as outlined above. For control purposes, age-matched C57Bl/6 mice that did not undergo any surgical procedure were injected intravenously with 125I-labeled CR2-Crry to confirm the specificity of CR2 targeting. After 12 hours of recovery, mice were sacrificed (n = 3 per group) and blood removed by cardiac puncture. Following blood removal, animals were perfused with PBS before removal of the heart, lung, liver, kidney, brain, and spinal cord. Tissues were rinsed in RPMI (Gibco, Carlsbad, CA) and counted with a Packard 5780 gamma counter at the 125I window with appropriate corrections for count decay.ResultsTime Course of Complement Activation after SCIThe presence of C3, deposition of which marks a site of complement activation by any pathway, was assessed in mice that had undergone SCI and in sham laminectomy controls. No staining for C3 was observed in sham operated-animals in any compartments of the spinal cord. In contrast, C3 deposition was evident following SCI in spinal cords harvested at 1 hour, 2 hours, 4 hours, 12 hours, and 24 hours after injury. At 1 hour, 2 hours, and 4 hours post-SCI, C3 deposition was centered to the white matter of the injury site and within the ventral horns of the gray matter (Figure 1A). At later time points of 12 hours and 24 hours, C3 staining was evident in surviving white matter, with staining also present throughout the gray matter and extending into the ventral and dorsal horns (Figure 1B). By day 3 after injury, complement deposition was almost undetectable, with no C3 staining evident at the injury site (Figure 1, C and D). This result is different from that reported for complement deposition in the rat spinal cord following injury, in which complement deposition was evident for up to 42 days after injury.6Anderson AJ Najbauer J Huang W Young W Robert S Upregulation of complement inhibitors in association with vulnerable cells following contusion-induced spinal cord injury.J Neurotrauma. 2005; 22: 382-397Crossref PubMed Scopus (21) Google Scholar An additional apparent difference in the mouse model was that, at all time points, spinal cord sections 10 mm rostral and caudal to the injury site showed a much reduced C3 staining pattern compared to sections taken from the injury site. Complement deposition was seen up to 20 mm from the injury site in rats, with no apparent decrease in immunoreactivity and with increasing distance from the site of injury.7Anderson AJ Robert S Huang W Young W Cotman CW Activation of complement pathways after contusion-induced spinal cord injury.J Neurotrauma. 2004; 21: 1831-1846Crossref PubMed Scopus (90) Google Scholar However, this apparent difference is likely a consequence of animal size and differences in size of impact injury required to produce an equivalent condition.Effect of C3 Deficiency and of Complement Inhibition on Locomotor Recovery following SCITo investigate the role of complement in SCI, we induced contusion injury to the spinal cord in wild-type mice and in mice deficient in C3, a central protein of the complement system and common for all pathways of activation. Following injury, locomotor recovery was assessed using the modification of the BBB rating scale.13Noble LJ Donovan F Igarashi T Goussev S Werb Z Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events.J Neurosci. 2002; 22: 7526-7535PubMed Google Scholar All animals had a BBB score of 21 pre-injury and a score of 0 immediately after injury, with bilateral hindlimb paralysis (Figure 2). Two days after injury, and every day thereafter through the termination of the study at day 21, the C3-deficient mice had a significantly improved BBB score compared to the wild-type controls (P < 0.001) (Figure 2). By day 21 after injury, the C3-deficient mice showed a near normal BBB score of 19.6 ± 1.2 (P < 0.001), whereas the BBB score for wild-type mice was only 11.5 ± 2.14 which was significantly lower than that of C3-deficient mice (P < 0.001). These data indicate that C3 plays an important role in the posttraumatic events that affect functional recovery. Next, we determined whether C3 blockade, using an intravenously administered inhibitor previously shown to target to sites of complement activation, is a feasible posttraumatic therapeutic approach for improving functional recovery.Figure 2Combined BBB locomotor scores post-SCI within sham, vehicle control, C3-deficient, and CR2-Crry groups. Note significant improvement in BBB score at day 3, 7, and 21 in both the C3-deficient and CR2-Crry groups when compared to vehicle controls (P = 0.001) (n = 12). The values are expressed as mean ± SE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Using the same spinal cord paradigm, a group of mice were treated with a single intravenous injection of 0.25 mg CR2-Crry at 1 hour after SCI. As with the C3-deficient mice, the CR2-Crry-treated mice had a significantly improved BBB score compared to sham-operated controls at all time points from day 2 following traumatic injury (P < 0.001) (Figure 2). The C3-deficient mice appeared to have a better outcome than the CR2-Crry-treated mice, but the difference was not significant.Effect of C3 Deficiency and of Complement Inhibition on the Extent of Tissue Destruction and Demyelination following SCITo determine whether C3 deficiency or complement inhibition with CR2-Crry attenuated overall spinal cord tissue damage, we determined the cross-sectioned area of spinal cords at 100-μm increments extending 2 mm either side of the initial injury impact site. Measurements were made using spinal cords isolated from C3-deficient mice and mice treated with CR2-Crry or vehicle control (PBS). At 24 hours after injury, the profile of tissue damage was similar in both C3-deficient and CR2-Crry-treated groups (Figure 3A). In the control group, there was a clear trend toward increased injury compared to the C3-deficient/inhibited groups, but by 24 hours after SCI the difference did not reach statistical significance at the injury site or on either side of the injury site. Comparable relative profiles were obtained for the three groups of animals at 72 hours after SCI (Figure 3B). Seven days after injury, however, there was significantly more tissue sparing at and around the injury site in C3-deficient mice and in mice treated with CR2-Crry compared to vehicle control mice (Figure 3C). There was no difference in tissue sparing between C3-deficient and CR2-Crry-treated mice at 7 days after SCI.Figure 3Tissue sparing as assessed by analyzing the cross-sectional area of spinal cords removed from vehicle controls, C3-deficient, and CR2-Crry-treated animals at 24 hours (upper panel), 72 hours (middle panel), and 7 days after injury (lower panel). Measurements were made from histological sections taken at 100-μm increments extending 2 mm either side of the injury site. No significant difference in tissue sparing was evident at 24 and 72 hours. Significant tissue sparing was noted in CR2-Crry and C3-deficient animals compared to vehicle controls at day 7 (P = 0.002). Mean ± SD, n = 4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also analyzed the extent of necrosis and demyelination in cords isolated from the different groups of animals 7 days and 21 days after SCI. In the vehicle control group, H&E staining of cord sections (centered around the injury site) revealed marked areas of necrosis with vacuolization of cells at day 7, with necrosis being somewhat less evident at day 21 (Figure 4, A and B). In contrast, the white matter beneath the injury site in cords isolated from C3-deficient mice appeared grossly intact at days 7 and 21 (Figure 4, C and D). Cords from CR2-Crry-treated mice also exhibited significant attenuation of injury when compared with vehicle controls, although there appeared to be more vacuolization in the cells within the white matter compared to the C3-deficient animals (Figure 4, E and F). Luxol fast blue staining of cord sections from the control group revealed obvious demylineation in the central core of the white matter beneath the impact site (Figure 5, A and B). By comparison, there was markedly less demyelination in cords from C3-deficient mice and CR2-Crry-treated mice at 7 and 21 days after SCI (Figure 5, C–F). There was no apparent difference in the extent of demyelination between the C3-deficient and complement-inhibited groups of animals.Figure 4H&E-stained sections of spinal cord centered on the injury site at days 7 and 21 after injury. A–B: vehicle control. C–D: C3-deficient animals. E–F: CR2-Crry-treated animals. Original magnification, ×100.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Luxol fast blue stained section of spinal cord centered on the injury site at days 7 and 21 after injury. A–B: vehicle control. C–D: C3-deficient animals. E–F: CR2-Crry-treated animals. Original magnification, ×100.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Neutrophil InfiltrationInfiltration of neutrophils is thought to be a significant factor in the development of secondary injury in spinal cords following traumatic injury, and complement activation products can provide activating and chemotactic signals and up-regulate expression of adhesion molecules. We therefore investigated the presence of neutrophils at the site of injury in animals from all three groups at 24 hours, 72 hours, and 7 days after injury. Neutrophil infiltration was most pronounced within the first 24 hours after injury in all groups, with declining numbers present at 72 hours and 7 days (Figure 6). At all time points, however, neutrophil infiltration was significantly inhibited in both C3-deficient and CR2-Crry-treated mice (P < 0.001). There was no significant difference between neutrophil numbers in the C3-deficient and the complement inhibited mice at any time point. In this experiment, the total number of neutrophils present on each section was counted. No distinction was made between white and gray matter, due to the extensive damage noted at 24 and 72 hours after injury and because sections were analyzed using immunofluorescence, which did not permit morphological evaluation.Figure 6Neutrophil infiltration as assessed by immunofluorescent staining with anti-mouse GR1-fluorescein labeled antibody. The total number of neutrophils per section was counted. The number of neutrophils is significantly lower in C3-deficient and CR2-Crry-treated animals across all time points when compared with vehicle control (* P = 0.001). Mean ± SD, n = 5 per group.View Large Image Figure ViewerDownload Hi-res