TY - JOUR
AU - Weaver, Lynne C.
AB - Abstract Spinal cord injury (SCI) provokes an inflammatory response that generates substantial secondary damage within the cord but also may contribute to its repair. Anti-inflammatory treatment of human SCI and its timing must be based on knowledge of the types of cells participating in the inflammatory response, the time after injury when they appear and then decrease in number, and the nature of their actions. Using post-mortem spinal cords, we evaluated the time course and distribution of pathological change, infiltrating neutrophils, monocytes/macrophages and lymphocytes, and microglial activation in injured spinal cords from patients who were ‘dead at the scene’ or who survived for intervals up to 1 year after SCI. SCI caused zones of pathological change, including areas of inflammation and necrosis in the acute cases, and cystic cavities with longer survival (Zone 1), mantles of less severe change, including axonal swellings, inflammation and Wallerian degeneration (Zone 2) and histologically intact areas (Zone 3). Zone 1 areas increased in size with time after injury whereas the overall injury (size of the Zones 1 and 2 combined) remained relatively constant from the time (1–3 days) when damage was first visible. The distribution of inflammatory cells correlated well with the location of Zone 1, and sometimes of Zone 2. Neutrophils, visualized by their expression of human neutrophil α-defensins (defensin), entered the spinal cord by haemorrhage or extravasation, were most numerous 1–3 days after SCI, and were detectable for up to 10 days after SCI. Significant numbers of activated CD68-immunoreactive ramified microglia and a few monocytes/macrophages were in injured tissue within 1–3 days of SCI. Activated microglia, a few monocytes/macrophages and numerous phagocytic macrophages were present for weeks to months after SCI. A few CD8+ lymphocytes were in the injured cords throughout the sampling intervals. Expression by the inflammatory cells of the oxidative enzymes myeloperoxidase (MPO) and nicotinamide adenine dinucleotide phosphate oxidase (gp91phox), and of the pro-inflammatory matrix metalloproteinase (MMP)-9, was analysed to determine their potential to cause oxidative and proteolytic damage. Oxidative activity, inferred from MPO and gp91phox immunoreactivity, was primarily associated with neutrophils and activated microglia. Phagocytic macrophages had weak or no expression of MPO or gp91phox. Only neutrophils expressed MMP-9. These data indicate that potentially destructive neutrophils and activated microglia, replete with oxidative and proteolytic enzymes, appear within the first few days of SCI, suggesting that anti-inflammatory ‘neuroprotective’ strategies should be directed at preventing early neutrophil influx and modifying microglial activation. inflammation, macrophage, neutrophil, oxidative activity, SCI Abbreviations Abbreviations β-APP β-amyloid precursor protein H & E haematoxylin and eosin H & E/LFB combined H & E and Luxol fast blue MMP matrix metalloproteinase MPO myeloperoxidase NADPH nicotinamide adenine dinucleotide phosphate SCI spinal cord injury Introduction Injury to the spinal cord provokes an inflammatory reaction that initially results in further tissue damage. Attenuation of the early inflammatory response to spinal cord injury (SCI) may therefore limit the extent of tissue injury and, accordingly, the consequent disability (Hall, 1992; Bethea, 2000; Gris et al., 2004). Hence, the development of effective acute therapies for SCI in humans requires a precise knowledge of the time course of entry of various inflammatory cells into the injured human spinal cord and activation of resident spinal cord cells such as microglia, and of their many actions, destructive or beneficial, that affect neural tissue. The mechanisms and time course of inflammation are well documented in animal models of SCI (Popovich et al., 1997; Schnell et al., 1999; Hausmann, 2003; Sroga et al., 2003). In rats, neutrophils appear at the primary lesion site 4–6 h after injury, peaking in number at 12–24 h and disappearing within ∼5 days (Taoka et al., 1997; Carlson et al., 1998). Oxidative and proteolytic enzymes produced by neutrophils sterilize the damaged area and prepare it for subsequent ‘repair’, but overwhelming numbers of neutrophils can cause ‘by-stander’ tissue damage (Taoka et al., 1997). Macrophages in the injured spinal cord are derived from blood-borne monocytes and resident microglia (seePopovich et al., 1999). Blood-borne monocyte/macrophages infiltrate the lesion 2 days after SCI in rats, achieve their highest density at 5–7 days, and persist for weeks to months (Popovich et al., 1997; Carlson et al., 1998). Microglia become activated within minutes to hours after SCI and are transformed into macrophages (Popovich et al., 2002). Macrophages in the injured rodent cord contribute to by-stander damage by releasing pro-inflammatory cytokines, reactive oxygen species, nitric oxide and proteases (Popovich et al., 1999; Popovich et al., 2002). In contrast to these destructive effects, they also participate in the removal of injured tissue debris and the release of protective cytokines that promote neuronal regeneration, wound healing and tissue repair (Rabchevsky and Streit, 1998; Schwartz, 2003). In animals, T-lymphocytes enter the injured spinal cord at different times, depending upon species and strain of animal (Sroga et al., 2003). T-lymphocytes are responsible for cell-mediated adaptive immunity (Friese and Fugger, 2005; Jones et al., 2005). Whether T-lymphocytes cause secondary degeneration or mediate wound repair after SCI remains highly controversial (Hauben and Schwartz, 2003; Friese and Fugger, 2005; Jones et al., 2005; Crutcher et al., 2006). The secondary spinal cord damage caused by neutrophils and macrophages in animal studies is caused in part by oxidative and proteolytic enzymes. Myeloperoxidase (MPO), a well-known oxidative enzyme, is expressed abundantly by neutrophils and other phagocytes (Taoka et al., 1997; Bao et al., 2004) and generates hypochlorous acid that kills pathogens, but also damages nearby tissue. Another damaging oxidative enzyme produced by inflammatory cells is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that generates the superoxide anion (Vaziri et al., 2004; Brandes and Kreuzer, 2005). The activity of this enzyme requires the catalytic subunit gp91phox on the cell membrane. gp91phox serves as an excellent marker of oxidative activity in the rat spinal cord (Bao et al., 2005). Inflammatory cells also release matrix metalloproteinases (MMPs), especially MMP-9, to permit them to penetrate the blood–CNS-barrier (Noble et al., 2002). MMP-9 is upregulated in leucocytes entering the spinal cord, directly facilitating their extravasation, and promoting the tissue damage that they cause (Mun-Bryce and Rosenberg, 1998; Wang et al., 2000; Noble et al., 2002). In contrast to the detailed information available from experimental animals, only a few studies of the inflammatory response to human SCI have been conducted and the very acute period after injury has rarely been assessed (Yang et al., 2004; Chang, 2006). Accordingly, we used immunohistochemical methods to evaluate the cellular inflammatory response to human SCI by determining the time course and distribution of neutrophil, mononuclear phagocyte and T-lymphocyte infiltration, as well as, microglial activation in post-mortem spinal cords with injury-to-death (‘survival’) intervals ranging from minimal [i.e. victims who ‘die at the scene’ (DAS)] to 1 year. Furthermore, we have assessed the potential of these inflammatory cells to contribute to secondary damage by investigating their expression of markers of oxidative stress, including the oxidative enzymes MPO and the catalytic subunit (gp91phox) of NADPH oxidase and of the pro-inflammatory protease MMP-9. Material and methods The Miami Project to Cure Paralysis provided spinal cords from 17 cases of SCI (2 females and 15 males with ages ranging from 6 to 88 years) and from 4 control cases (4 males, 21–86 years) who had sustained vertebral fractures and/or traumatic head injuries but had no structural evidence of SCI. The hospitals of the London Health Science Centre provided injured spinal cords from 11 cases (6 females, 5 males, 13–82 years). More than one spinal cord segment (i.e. cervical and thoracic) was used from each control case. Survival times after injury ranged from minimal (i.e. patients who died at the scene of the accident) to 1 year (Table 1). The circumstances of SCI, patient demographic information and details of the general and neuropathological post-mortem assessment for each case were obtained from autopsy reports. Patients with infectious and inflammatory complications not directly associated with trauma, such as sepsis, were excluded from the study because the effect of concurrent systemic inflammation on the intraspinal inflammatory response is unknown. The spinal cord injuries were classified on the basis of their histological appearance as ‘contusion/cyst’ (n = 17), ‘massive compression’ (n = 7) or ‘laceration’ (n = 4) (Bunge et al., 1993; Shkrum and Ramsay, 2006). Contusional injuries were characterized by an intact pia and relative preservation of the anatomical relations of various elements of the spinal cord, and variable degrees of injury ranging from involvement of the entire cross-sectional area to large usually asymmetric areas of tissue damage. In older injuries, cyst-like areas were present. Massive compression injuries were characterized by disruption of the pia and severe distortion and disruption of spinal cord parenchyma. Laceration injuries, which by definition were perforating or penetrating injuries caused by weapons or projectiles, were associated with breaching of the pia and linear tearing of the cord tissue. Table 1 Descriptions of spinal cord injury cases Case number . Survival . Age . Sex . Nature of accident . Spinal level(s) involved . Type of injury . Source . 521 0 h DAS 63 M MVA C M LHSC 623 0 h DAS 53 M MVA T4 C/C LHSC 349 0 h DAS 23 M MVA T M LHSC 144 1 h 6 M Ped. versus car C2, T6-7 C/C MP 155 1 h 35 M GSW T7-12 C/C MP 130 2 h 55 F MVA T6-7 L LHSC 05 3 h 19 M GSW T12-L2 L MP 48 4 h 72 M Fall T5-7 C/C MP 488 1 day 13 F Ped. versus car C6-7 C/C LHSC 201 1 day 82 F Fall C4 C/C LHSC 119 2 days 37 M Altercation C3-4 M MP 162 3 days 84 M Fall C3-4 M MP 176 3 days 73 M MVA C3 C/C MP 120 5 days 88 M MVA C8 C/C MP 175 5 days 33 M MVA T2-3 C/C MP 206 5 days 16 M MVA T3-8 C/C MP 20 8 days 20 F GSW C6 L MP 181 10 days 65 M Fall C6 M MP 66 10 days 22 M Diving C6-8 C/C MP 58 2 weeks 80 M Fall C7 C/C MP 208 3 weeks 57 F Fall C7 C/C LHSC 378 3 weeks 47 M MVA T4-5 L LHSC 368 5 weeks 23 F MVA C5-7 C/C LHSC 96 6 weeks 67 M Fall C5-7 C/C MP 408 2 months 35 F MVA T C/C LHSC 47 ∼6 months 24 M MVA C6-7 M LHSC 94 7 months 43 M MVA C4-6 M MP 29 1 year 45 F MVA C7 C/C MP Controls* 146 1.5 h 21 M Bicycle versus car C4 n/a MP 131 5 h 26 M MVA C8, T1 n/a MP 184 4.5 days 86 M MVA T11, L3 n/a MP 159 5 days 28 M MVA C8, T9 n/a MP Case number . Survival . Age . Sex . Nature of accident . Spinal level(s) involved . Type of injury . Source . 521 0 h DAS 63 M MVA C M LHSC 623 0 h DAS 53 M MVA T4 C/C LHSC 349 0 h DAS 23 M MVA T M LHSC 144 1 h 6 M Ped. versus car C2, T6-7 C/C MP 155 1 h 35 M GSW T7-12 C/C MP 130 2 h 55 F MVA T6-7 L LHSC 05 3 h 19 M GSW T12-L2 L MP 48 4 h 72 M Fall T5-7 C/C MP 488 1 day 13 F Ped. versus car C6-7 C/C LHSC 201 1 day 82 F Fall C4 C/C LHSC 119 2 days 37 M Altercation C3-4 M MP 162 3 days 84 M Fall C3-4 M MP 176 3 days 73 M MVA C3 C/C MP 120 5 days 88 M MVA C8 C/C MP 175 5 days 33 M MVA T2-3 C/C MP 206 5 days 16 M MVA T3-8 C/C MP 20 8 days 20 F GSW C6 L MP 181 10 days 65 M Fall C6 M MP 66 10 days 22 M Diving C6-8 C/C MP 58 2 weeks 80 M Fall C7 C/C MP 208 3 weeks 57 F Fall C7 C/C LHSC 378 3 weeks 47 M MVA T4-5 L LHSC 368 5 weeks 23 F MVA C5-7 C/C LHSC 96 6 weeks 67 M Fall C5-7 C/C MP 408 2 months 35 F MVA T C/C LHSC 47 ∼6 months 24 M MVA C6-7 M LHSC 94 7 months 43 M MVA C4-6 M MP 29 1 year 45 F MVA C7 C/C MP Controls* 146 1.5 h 21 M Bicycle versus car C4 n/a MP 131 5 h 26 M MVA C8, T1 n/a MP 184 4.5 days 86 M MVA T11, L3 n/a MP 159 5 days 28 M MVA C8, T9 n/a MP C/C = contusion/cyst type injuries; M = massive compression type injuries; L = laceration type injuries; LHSC = London Health Sciences Center; MP = Miami Project to Cure Paralysis; DAS = dead at the scene; MVA = motor vehicle accident; GSW = gunshot wound; Ped. = pedestrian. *Sections were taken from control cords in segments that correspond to segments in the injured cords. Open in new tab Table 1 Descriptions of spinal cord injury cases Case number . Survival . Age . Sex . Nature of accident . Spinal level(s) involved . Type of injury . Source . 521 0 h DAS 63 M MVA C M LHSC 623 0 h DAS 53 M MVA T4 C/C LHSC 349 0 h DAS 23 M MVA T M LHSC 144 1 h 6 M Ped. versus car C2, T6-7 C/C MP 155 1 h 35 M GSW T7-12 C/C MP 130 2 h 55 F MVA T6-7 L LHSC 05 3 h 19 M GSW T12-L2 L MP 48 4 h 72 M Fall T5-7 C/C MP 488 1 day 13 F Ped. versus car C6-7 C/C LHSC 201 1 day 82 F Fall C4 C/C LHSC 119 2 days 37 M Altercation C3-4 M MP 162 3 days 84 M Fall C3-4 M MP 176 3 days 73 M MVA C3 C/C MP 120 5 days 88 M MVA C8 C/C MP 175 5 days 33 M MVA T2-3 C/C MP 206 5 days 16 M MVA T3-8 C/C MP 20 8 days 20 F GSW C6 L MP 181 10 days 65 M Fall C6 M MP 66 10 days 22 M Diving C6-8 C/C MP 58 2 weeks 80 M Fall C7 C/C MP 208 3 weeks 57 F Fall C7 C/C LHSC 378 3 weeks 47 M MVA T4-5 L LHSC 368 5 weeks 23 F MVA C5-7 C/C LHSC 96 6 weeks 67 M Fall C5-7 C/C MP 408 2 months 35 F MVA T C/C LHSC 47 ∼6 months 24 M MVA C6-7 M LHSC 94 7 months 43 M MVA C4-6 M MP 29 1 year 45 F MVA C7 C/C MP Controls* 146 1.5 h 21 M Bicycle versus car C4 n/a MP 131 5 h 26 M MVA C8, T1 n/a MP 184 4.5 days 86 M MVA T11, L3 n/a MP 159 5 days 28 M MVA C8, T9 n/a MP Case number . Survival . Age . Sex . Nature of accident . Spinal level(s) involved . Type of injury . Source . 521 0 h DAS 63 M MVA C M LHSC 623 0 h DAS 53 M MVA T4 C/C LHSC 349 0 h DAS 23 M MVA T M LHSC 144 1 h 6 M Ped. versus car C2, T6-7 C/C MP 155 1 h 35 M GSW T7-12 C/C MP 130 2 h 55 F MVA T6-7 L LHSC 05 3 h 19 M GSW T12-L2 L MP 48 4 h 72 M Fall T5-7 C/C MP 488 1 day 13 F Ped. versus car C6-7 C/C LHSC 201 1 day 82 F Fall C4 C/C LHSC 119 2 days 37 M Altercation C3-4 M MP 162 3 days 84 M Fall C3-4 M MP 176 3 days 73 M MVA C3 C/C MP 120 5 days 88 M MVA C8 C/C MP 175 5 days 33 M MVA T2-3 C/C MP 206 5 days 16 M MVA T3-8 C/C MP 20 8 days 20 F GSW C6 L MP 181 10 days 65 M Fall C6 M MP 66 10 days 22 M Diving C6-8 C/C MP 58 2 weeks 80 M Fall C7 C/C MP 208 3 weeks 57 F Fall C7 C/C LHSC 378 3 weeks 47 M MVA T4-5 L LHSC 368 5 weeks 23 F MVA C5-7 C/C LHSC 96 6 weeks 67 M Fall C5-7 C/C MP 408 2 months 35 F MVA T C/C LHSC 47 ∼6 months 24 M MVA C6-7 M LHSC 94 7 months 43 M MVA C4-6 M MP 29 1 year 45 F MVA C7 C/C MP Controls* 146 1.5 h 21 M Bicycle versus car C4 n/a MP 131 5 h 26 M MVA C8, T1 n/a MP 184 4.5 days 86 M MVA T11, L3 n/a MP 159 5 days 28 M MVA C8, T9 n/a MP C/C = contusion/cyst type injuries; M = massive compression type injuries; L = laceration type injuries; LHSC = London Health Sciences Center; MP = Miami Project to Cure Paralysis; DAS = dead at the scene; MVA = motor vehicle accident; GSW = gunshot wound; Ped. = pedestrian. *Sections were taken from control cords in segments that correspond to segments in the injured cords. Open in new tab In all cases, tissue samples from the centre of SCI and at various distances above and below the injury were obtained. The data from tissue from the centre of the lesion were used to compare the inflammatory responses between cases whereas those from the remote, uninjured segments of the spinal cord served as within-case controls. Between-case comparison of the remote samples was not possible because, for different cases, the distance of these samples from the lesion centre was variable. All tissue samples had been removed within 24 h of death and fixed in neutral buffered formalin for several weeks. Blocks from the spinal cords were dehydrated, embedded in paraffin wax, cut into sets of 6 μm thick sections and placed on positively charged glass slides. One set of sections was stained with haematoxylin-eosin (H&E), and the remaining sets were used for immunohistochemistry. A subset of sections from four cords within each injury-to-death interval was stained with H & E/Luxol fast blue (H&E/LFB). A second subset of these sections was stained with Bielschowsky's silver stain. Immunohistochemistry The antibacterial protein family α-defensins-1-3 (subsequently termed ‘defensin’) was used as a highly selective marker of neutrophils (Schluesener and Meyermann, 1995; Barnathan et al., 1997; Agerberth et al., 2000) and was labelled by anti-NCL-Defensin antibody (HNP 1–3) (1:1000, Novocastra Laboratories Ltd., Newcastle, UK). The remaining cells and enzymes were identified by the following antibodies: anti-β-amyloid precursor protein (β-APP) (1:4000, Chemicon International), labelling damaged axons; anti-PG-M1 (1:1000, Dako, Glostrup, Denmark), directed against CD68, a lysosomal protein expressed by phagocytic macrophages of microglial and monocytic origin (Greaves et al., 1998; van den Berg et al., 2001); anti-CD8α (1:100, Dako, Glostrup, Denmark), labelling cytotoxic T and natural killer cells; anti-CD4 (1:100, Novocastra Laboratories Ltd., Newcastle, UK), labelling helper/regulator T cells; anti-CD20cy (1:100, Dako, Glostrup, Denmark), labelling B cells; anti-MPO (1:10 000, Dako, Glostrup, Denmark) recognizing MPO; anti-gp91phox, labelling the active conformation of NADPH oxidase (1:500, Upstate Biotechnology, Lake Placid, NY, USA); and anti-MMP-9 (1:1000, Chemicon International, Temecula, CA, USA), labelling inactive and activated forms of MMP-9. The sections used for immunohistochemistry were dried at 37°C overnight, deparaffinized in xylene, and rehydrated through a series of graded ethyl alcohols. Endogenous peroxidase was inactivated by treatment with 3% H2O2 in 100% methanol for 5 min. Following a 5 min wash in phosphate-buffered saline (PBS, pH 7.4), microwave heat-induced antigen retrieval in 10 mmol/l citrate buffer, pH 6.0, was carried out. Non-specific staining was blocked by incubating the sections in 10% normal horse serum for 1 h at room temperature. Afterwards, the sections were incubated with the various primary antibodies at room temperature for 48 h in 2% normal horse serum in a humidified chamber. Sections were then washed in PBS and incubated overnight in either a donkey anti-mouse secondary antibody conjugated to biotin or a donkey anti-rabbit secondary antibody conjugated to biotin (both at 1:1000, both from Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) in 2% normal horse serum. Next, sections were washed in PBS and incubated with extravidin peroxidase (1:1500, Sigma, St Louis, MO, USA) in 2% normal horse serum for 4 h. Tissues were then incubated for 7 min with the chromogen diaminobenzidine (DAB) and glucose oxidase (both from Sigma, St Louis, MO, USA) for visualization of antibody binding. Following PBS washes, the sections were counterstained with Gill III haematoxylin, dehydrated through ascending ethyl alcohols, cleared in xylene, and cover-slipped with DPX mountant. Sections from surgical specimens of acutely inflamed appendices (in which numerous neutrophils and abundant lymphoid follicles are present) were exposed to each of the primary antibodies, to serve as positive controls for immunolabelling of neutrophils, monocytes/macrophages and lymphocytes. Negative controls included sections in which the primary antibody was omitted, and sections incubated with isotype-matched antibodies (1:100–1:10 000, IgG). These positive and negative controls were processed with every batch of immunohistochemical slides. Quantification of the area of necrosis at the lesion epicentre The cord injury was assessed microscopically, using brightfield optics, by examining one H&E- or H&E/LFB-stained section from the lesion centre of each case or from cervical, thoracic and lumbar sections from control cases. The entire cross-sectional area of the section was digitized using a Retiga 1300 videocamera (Quantitative Imaging Corporation, Burnaby, BC, Canada), and ImagePro Plus software (Media Cybernetics, Silver Spring, MD, USA) as described below. Using the quantification software, calibrated as described below, the area of necrosis (areas in which the normal staining intensity of the tissues and the capacity to distinguish histologically between various cytological elements were lost), in the short survival cases, and the cystic regions, in the longer survival cases, in each photomontage were selected by the experimenter. These areas will be defined as Zone 1 in Results. The cumulative area occupied by the pixels in the area selected was determined and expressed as a percentage of the ‘area of interest’ (AOI), which in this case, was the entire cross-sectional area of the spinal cord, excluding the pia. Semi-quantitative assessment of overall injury To assess the overall injury, each spinal cord cross-section was divided into eight distinct areas (left and right ventral horns and left and right dorsal, ventral and lateral funiculi—the posterior horns are too narrow to allow reliable assessment) and the overall injury in each of these areas was scored as: 0 = no injury; 1 = trace injury; 2 = slight injury (injury occupies one-quarter of the region); 3 = moderate injury (injury occupies half of the region); 4 = moderately severe injury (injury occupies three-quarters of the region); and 5 = severe injury (injury occupies all of the region). The presence of scant petechiae defined ‘trace’ injury whereas ‘injury’ in all other categories included, in various combinations, extensive bleeding, necrosis (identified by tissue pallor, loss of cellular architecture and cystic cavities), ‘oedema’ (characterized by tissue microvacuolation) and axonal swelling. The overall injury score is a summation of the pathological changes that will be defined as Zones 1 and 2 in Results. This assessment of injury did not show a predilection in terms of severity or location for any of the eight grey or white matter regions. Therefore the average score for the grey matter regions and for the white matter regions was calculated. Quantification of neutrophil (defensin), microglia/macrophage (CD68) and MPO immunolabelling A section of the spinal cord at the centre of the injury of each case was viewed through a ×4 objective. Digital photographs of 20–24 individual fields of view, covering the entire cross-sectional area of the spinal cord, were recorded digitally using a Retiga 1300 videocamera (Quantitative Imaging Corporation, Burnaby, BC, Canada), and ImagePro Plus software (Media Cybernetics, Silver Spring, MD, USA). The individual images were then ‘stitched’ together electronically by ImagePro Plus (Fig. 1A–C). Afterwards, using the quantification software, the area represented by one pixel was calibrated in μm2 for the ×4 objective and a colour-based threshold detection function was applied to each photomontage by the experimenter to select the brown intracellular DAB staining, excluding the blue haematoxylin-stained nuclei and any extracellular staining or background (Fig. 1B–E). The cumulative area occupied by these pixels was determined and expressed as a percentage of the entire cross-sectional area of the spinal cord, excluding the pia (AOI). This method was used to quantify separately the inflammatory response attributable to neutrophils or microglia/macrophages. The area of immunoreactivity assessed by this method gives an overall estimate of the ‘inflammatory response’ associated with a given cell type (Popovich et al., 1997; Saville et al., 2004). The extent of immunoreactivity reflects the number of cells involved in the inflammatory response and their size. Cell size is likely to be a significant part of the measured macrophage and microglia response, since activation of these cells causes them to enlarge, but, in the case of neutrophils, the area of immunoreactivity will be more closely correlated with the numbers of neutrophils because these cells shrink as they involute. We chose this method instead of cell counting because neutrophils, microglia and macrophages may be impossible to distinguish on cytological criteria when the entire cell (such as a ramified microglia) is not apparent in a section and or when cells cluster. The areas of defensin immunoreactivity in grey and white matter were compared to assess any differences between these areas in the neutrophil inflammatory response. In an attempt to confirm the relative specificity of defensin and MPO immunolabelling for neutrophils, areas of MPO immunolabelling were measured in four cases selected from the 1–3 day interval and in four cases from the 5–10 day interval and compared with corresponding areas occupied by defensin-ir cells (i.e. neutrophils) or CD68-ir cells (i.e. monocytes/macrophages; Table 3). Fig. 1 Open in new tabDownload slide Method for analysis of areas of immunoreactivity for defensin and CD68 in spinal cord sections. (A) Gross morphology of spinal cord of a typical 1–3 day survival interval case (H & E). (B and D) The immunoreactivity for defensin, indicating neutrophil infiltration, at low and high magnification. (C and E) The colour-based threshold detection system for quantification selects areas of immunoprecipitate of intensity greater than background and marks them with a red mask. Note that the areas of immunoreactivity analysed are exclusively intracellular. Scale bars: (A–C) 2.5 mm; (D and E) 50 μm. Fig. 1 Open in new tabDownload slide Method for analysis of areas of immunoreactivity for defensin and CD68 in spinal cord sections. (A) Gross morphology of spinal cord of a typical 1–3 day survival interval case (H & E). (B and D) The immunoreactivity for defensin, indicating neutrophil infiltration, at low and high magnification. (C and E) The colour-based threshold detection system for quantification selects areas of immunoprecipitate of intensity greater than background and marks them with a red mask. Note that the areas of immunoreactivity analysed are exclusively intracellular. Scale bars: (A–C) 2.5 mm; (D and E) 50 μm. Quantification of CD8+cells The relative abundance of CD8+cells in these cases could not be assessed by measuring areas of immunoreactivity due to the paucity of these cells in the spinal cords. Instead, they were counted in four randomly selected high power (×40) fields of view and an average number per field was generated for each case. Morphological characterization of microglia and macrophages and expression of enzymes Phagocytic microglia and monocyte/macrophages were immunoreactive for CD68. CD68-labelled microglia with discrete thin processes were termed ‘ramified microglia’ and were considered to be microglia in the earliest state of activation. ‘Activated microglia’ refers to those with hypertrophic cell bodies and shorter, thicker cell processes, indicating transformation into macrophages. Macrophages were defined as large rounded cells (>20 μm in diameter) with large clear cytoplasmic vacuoles and inclusions that indicated ongoing or previous phagocytic activity. Currently, no available techniques can distinguish between phagocytic macrophages derived from microglia or from haematogenous monocyte/macrophages. The expression of MPO and gp91phox, markers of oxidative reactivity, and MMP-9, a pro-inflammatory protease, was examined at each of the time intervals after injury (intervals are defined in Results). We used cellular morphology and serial sections stained for defensin and CD68 in each case to identify the cell populations with immunoreactivity for MPO, gp91phox and MMP-9. Despite the clustering of cells, a sufficient number were separate enough to permit their morphology to be examined. When only a fraction of a population of a specific cell type expressed a given protein, the percentage of cells expressing it in five ×40 fields was determined in each spinal cord. When ramified microglia were in a ‘resting state’, they did not express CD68 and were not readily visible. Therefore, we had no measure of the entire population of ramified microglia from which to determine the proportion expressing proteins such as MPO or gp91phox. We instead described the approximate numbers of MPO- or gp91phox-ir cells, with morphology of ramified microglia, found in ×40 microscopic fields. Statistical analysis Data were subjected to parametric statistical analysis using completely randomized one-way ANOVA (Snedecor and Cochran, 1989). Fisher's LSD protected t-test was used to determine differences between mean values. The probability value required to attain statistical significance was P < 0.05. In one analysis a one-tailed Student's t-test was used to determine the difference between mean values (P < 0.05). Spearman's correlation analysis was used to determine the relationship between time after injury and the relative area of necrosis at the lesion epicentre (P < 0.05). Results These data are organized by inflammatory cell type and the cases are categorized into the following five groups: no SCI and 0–4 h, 1–3 days, 5–10 days, and weeks to months, up to 1 year after injury. Zonal distribution of injury SCI resulted in a zonal distribution of pathological changes at the lesion site in cords at intervals >4 h after injury (Fig. 2A). The relative sizes of these zones varied substantially from case to case. An area of intense tissue injury, either in the form of necrosis (in the shorter survival cases; Fig. 2B and C left) or as cystic change (in the longer surviving cases; Fig. 2D–Fleft), was often, but not always, centred on the grey matter (Zone 1, outlined in green on Fig. 2B–Fleft). An exception to this general pattern is the cord at 2 weeks that had selective necrosis of the white matter (Fig. 2D). The areas of necrosis and cystic cavities contained, or were surrounded by, inflammatory cells such as neutrophils, labelled by their expression of defensin, (Fig. 2Bright) and macrophages, labelled by their expression of CD68, (Fig. 2C–Fright). Areas of incomplete injury (Zone 2) were located around the perimeter of Zone 1 and in more widely but haphazardly distributed locations outside and mutually exclusive of Zone 1. The changes in Zone 2 included axonal injury, Wallerian degeneration and inflammatory cells (neutrophils and activated microglia) in the cases that survived 1–3 or 5–10 days after injury, and residual axonal degeneration, progressive gliosis and variable numbers of macrophages in cases that survived weeks to months after injury. Small regions of apparently intact grey matter and white matter (Zone 3) lay adjacent to areas containing Zone 2 changes. In the very acutely (0–4 h) injured spinal cords, the patterns of zone formation had not developed. Fig. 2 Open in new tabDownload slide Histopathological changes at the lesion site. (A) Sketch illustrating the typical zonal distribution of pathological changes at the lesion epicentre (Zone 1 = area of intense tissue injury characterized by necrosis in cases with short survival and cystic cavities in cases with longer survival; Zone 2 = area surrounding Zone 1 containing ‘incomplete’ tissue injury; Zone 3 = area of intact grey and/or white matter). (B–F) Photomontages of spinal cord sections stained (left column) with H&E/LFB or H&E and (right column) corresponding adjacent sections treated with (B) defensin immunohistochemistry to visualize neutrophils or (C–F) CD68 immunohistochemistry to visualize macrophages. ‘Zone 1’ is outlined in green in the left columns. Spinal cords at Day 1, 6th cervical segment (1 d C6) and 10 days, 6th cervical segment (10 d C6) have clear necrosis, predominantly in the grey matter, whereas cords at 2 weeks, 7th cervical segment (2 wk C7), 3 weeks, 8th cervical segment (3 wk C8) and at 6 months, 6th cervical segment (6 mo C6) show cavity formation. Note the spatial relationship between the areas of necrosis or cystic change and the distribution of the inflammatory cells. Scale bar: 2.5 mm, applicable to all photomontages. (G) Sections stained with H&E/LFB reveal petechiae (arrow) in the grey matter and tissue fragmentation (arrowhead) 1 h after injury (scale bar: 100 μm), and (H) swirling malorientation of the fibre tracts in the posterior column from the same case, compared with the (I) normally oriented, transversely cut posterior column fibre tracts from an uninjured spinal cord. Scale bar (50 μm) on panel (I) applies to (H). (J) One day after injury, spinal cord tissue was extensively fragmented and necrotic (H&E), and contained localized haemorrhage (asterisk) (scale bar = 1 mm), (K) dense grey matter necrosis (H&E), and (L) neutrophil infiltration associated with loose necrotic tissue (H&E; arrows, representative neutrophils). Scale bar (50 μm) on panel (L) applies to (K). (M–O) Axonal swellings and associated vacuolation of white matter 3 days after injury demonstrated with (M) H&E/LFB staining (arrows, axonal swellings; asterisk, vacuolation), (N) Bielschowsky's method (green arrow = granular argyrophilic axonal swelling; red arrow = axonal swelling with no argyrophilic material) and (O) β-APP immunostaining (arrow = β-APP-immunopositive axonal swelling; asterisk = vacuolation). Scale bar (50 μm) on panel (O) applies to (M and N). (P) Anterior horn cells adjacent to areas of necrosis have hypereosinophilic cytoplasm (arrowhead), perineuronal vacuolation (asterisk), and normal blue LFB-stained lipofuscin (arrow) 10 days after injury. Scale bar: 50 μm. (Q) The intensity of myelin staining, shown by H&E/LFB, and (R) the axonal density, shown by Bielschowsky's method, decline gradually and proportionally as the lesion epicentre (left) is approached. Arrows indicate region from which insets were taken. Scale bar (100 μm) and inset scale bar (25 μm) on panel R applies to (Q). (S) Scatter plot demonstrating relative areas of Zone 1 areas of necrosis or cyst formation (ordinate, expressed as % of total section area) calculated for each case, plotted against injury-to-death intervals (abscissa) for the control group (n = 3) and injury groups (n = 22). Analysis was performed on H&E- or H&E/LFB-stained sections. The relative size of Zone 1 necrosis or cyst formation increased with time after injury (Spearman correlation, r = 0.7781, P < 0.00001). (T) Pooled overall injury scores for the control group and the different survival interval groups (means ± SEM for grey and white matter: asterisk indicates significantly different with uninjured controls and with 0–4 h after injury (grey matter, one-way ANOVA, F4,26 = 8.79, P = 0.0001, Fisher's protected t-test, P < 0.01; white matter, one-way ANOVA, F4,27 = 6.89, P = 0.0006, Fisher's protected t-test, P < 0.01). The severity of injuries did not differ between the 1–3 and 5–10 days, and weeks-to-months groups. Fig. 2 Open in new tabDownload slide Histopathological changes at the lesion site. (A) Sketch illustrating the typical zonal distribution of pathological changes at the lesion epicentre (Zone 1 = area of intense tissue injury characterized by necrosis in cases with short survival and cystic cavities in cases with longer survival; Zone 2 = area surrounding Zone 1 containing ‘incomplete’ tissue injury; Zone 3 = area of intact grey and/or white matter). (B–F) Photomontages of spinal cord sections stained (left column) with H&E/LFB or H&E and (right column) corresponding adjacent sections treated with (B) defensin immunohistochemistry to visualize neutrophils or (C–F) CD68 immunohistochemistry to visualize macrophages. ‘Zone 1’ is outlined in green in the left columns. Spinal cords at Day 1, 6th cervical segment (1 d C6) and 10 days, 6th cervical segment (10 d C6) have clear necrosis, predominantly in the grey matter, whereas cords at 2 weeks, 7th cervical segment (2 wk C7), 3 weeks, 8th cervical segment (3 wk C8) and at 6 months, 6th cervical segment (6 mo C6) show cavity formation. Note the spatial relationship between the areas of necrosis or cystic change and the distribution of the inflammatory cells. Scale bar: 2.5 mm, applicable to all photomontages. (G) Sections stained with H&E/LFB reveal petechiae (arrow) in the grey matter and tissue fragmentation (arrowhead) 1 h after injury (scale bar: 100 μm), and (H) swirling malorientation of the fibre tracts in the posterior column from the same case, compared with the (I) normally oriented, transversely cut posterior column fibre tracts from an uninjured spinal cord. Scale bar (50 μm) on panel (I) applies to (H). (J) One day after injury, spinal cord tissue was extensively fragmented and necrotic (H&E), and contained localized haemorrhage (asterisk) (scale bar = 1 mm), (K) dense grey matter necrosis (H&E), and (L) neutrophil infiltration associated with loose necrotic tissue (H&E; arrows, representative neutrophils). Scale bar (50 μm) on panel (L) applies to (K). (M–O) Axonal swellings and associated vacuolation of white matter 3 days after injury demonstrated with (M) H&E/LFB staining (arrows, axonal swellings; asterisk, vacuolation), (N) Bielschowsky's method (green arrow = granular argyrophilic axonal swelling; red arrow = axonal swelling with no argyrophilic material) and (O) β-APP immunostaining (arrow = β-APP-immunopositive axonal swelling; asterisk = vacuolation). Scale bar (50 μm) on panel (O) applies to (M and N). (P) Anterior horn cells adjacent to areas of necrosis have hypereosinophilic cytoplasm (arrowhead), perineuronal vacuolation (asterisk), and normal blue LFB-stained lipofuscin (arrow) 10 days after injury. Scale bar: 50 μm. (Q) The intensity of myelin staining, shown by H&E/LFB, and (R) the axonal density, shown by Bielschowsky's method, decline gradually and proportionally as the lesion epicentre (left) is approached. Arrows indicate region from which insets were taken. Scale bar (100 μm) and inset scale bar (25 μm) on panel R applies to (Q). (S) Scatter plot demonstrating relative areas of Zone 1 areas of necrosis or cyst formation (ordinate, expressed as % of total section area) calculated for each case, plotted against injury-to-death intervals (abscissa) for the control group (n = 3) and injury groups (n = 22). Analysis was performed on H&E- or H&E/LFB-stained sections. The relative size of Zone 1 necrosis or cyst formation increased with time after injury (Spearman correlation, r = 0.7781, P < 0.00001). (T) Pooled overall injury scores for the control group and the different survival interval groups (means ± SEM for grey and white matter: asterisk indicates significantly different with uninjured controls and with 0–4 h after injury (grey matter, one-way ANOVA, F4,26 = 8.79, P = 0.0001, Fisher's protected t-test, P < 0.01; white matter, one-way ANOVA, F4,27 = 6.89, P = 0.0006, Fisher's protected t-test, P < 0.01). The severity of injuries did not differ between the 1–3 and 5–10 days, and weeks-to-months groups. Histopathological characterization In the uninjured control cases, no histopathological abnormalities were identified. Similarly, no histopathological abnormalities were identified in the DAS cases, with the exception of rare petechiae in the grey matter. The spinal cords from cases that died 1–4 h after injury contained various degrees of tissue fragmentation, petechiae (particularly in the grey matter) (Fig. 2G), a haphazard, swirling malorientation of the fibre tracts in the funiculi (Fig. 2H versus I) and no abnormalities of β-APP immunolabelling (data not shown). The true extent of injury or areas where necrosis would have appeared, had the individual survived, could not be predicted from the distribution of abnormalities in this tissue. Malorientation of the fibres in the funiculi and fragmentation of the tissue were still evident 1–3 days after SCI (Fig. 2J). Petechiae were more prominent and often confluent. Necrosis (loss of staining intensity and cytological definition, as defined in Material and methods) was established usually in one confluent area, although some spinal cords had separate areas of necrosis. The necrotic areas tended to be haemorrhagic (Fig. 2J) and they were pronounced in, and sometimes limited to, grey matter (Fig. 2K). These areas of necrosis are defined as Zone 1 (Fig. 2A). Neutrophils were evident around the margins of the necrotic areas and, in one case, these inflammatory cells were associated with a distinct loosening of tissue texture (Fig. 2L). In an otherwise intact adjacent grey matter, changes in the anterior horn cells, included lysis of the Nissl substance, intensification of cytoplasmic eosinophilia, perineuronal vacuolation and karyorhexis or vesicular nuclear change, representing ischaemic neuronal necrosis, chromatolysis or both (Zone 2 change, seeFig. 2A). Focal white matter vacuolation and axonal swelling in various degrees were evident at the margins of the necrosis and scattered haphazardly through the white matter (Fig. 2M). Axonal swellings were incompletely filled with argyrophilic material (Fig. 2N), and solitary or discrete groups of axonal swellings were strongly β-APP-immunopositive (Fig. 2O). These are examples of the types of pathological changes defined as Zone 2. The distribution of the pathological changes at 5–10 days after injury resembled the distribution in cords at 1–3 days. However, at 5–10 days the necrotic areas contained abundant macrophages (Fig. 2C–F) and the adjacent, better preserved tissues (Zone 2), contained numerous and widely distributed axonal swellings with strong argyrophilia and a variable intensity of β-APP-immunopositivity (data not shown). The intensity of myelin staining was proportional to the argyrophilic axonal labelling, and provided no qualitative evidence of demyelination (data not shown). However, demyelination was difficult to detect against the background of the varied axonal and myelin changes that were present in this time interval and, in the limited numbers of sections available for this study, demyelination could not be confirmed or excluded. The anterior horn cells adjacent to areas of necrosis resembled those in the 1–3 days time period (Fig. 2P). In spinal cords at weeks to months after injury, areas of necrosis contained abundant macrophages or, in the longer survival cases, various degrees of cyst formation occurred (Fig. 2D–F). The distribution of these cysts was reminiscent of the distribution of necrosis in cases with shorter survival (1–10 days). In one case of extensive necrosis, a central area of ‘mummified’ necrosis (Zone 1) was surrounded by a rich mantle of macrophages in Zone 2 (Fig. 2E). The better preserved areas of the spinal cord (Zone 2) in the cases surviving for a few weeks contained macrophages, plump reactive gemistocytic astrocytes and numerous β-APP-immunopositive and argyrophilic axonal swellings (not shown). Many of the axonal swellings in these cases were β-APP-immunonegative. Fine gliovascular septa bridged the cystic spaces and a fine, dense network of fibrillary astrocytic processes surrounded the cystic areas in cases surviving for several months (data not shown). In one case with extensive spinal cord cystic change, the cystic area contained numerous peripheral nerve bundles and mild pial and perivascular fibrosis. Some gradation in intensity of myelin staining occurred at margins of the necrotic areas (Fig. 2Q), ranging from light, close to the lesion, to normal, approximately a millimetre away, but, in these areas, the numbers of argyrophilic axons were also proportionally graded with distance from the lesion (Fig. 2R). The areas of necrosis and/or cysts (Zone 1) within lesion epicentres (expressed as % of total section area) were measured for each case (Fig. 2S) and plotted against the injury-to-death intervals for the control group (n = 3) and injury groups (n = 22). Analysis was performed on H & E or H & E/LFB-stained sections. The relative size of Zone 1 in the spinal cords increased significantly with time after injury (Spearman correlation, r = 0.7781, P < 0.0001). The semi-quantitative assessment of overall tissue damage in Zones 1 and 2 combined (overall injury, see Material and methods) in grey and white matter, revealed no significant histopathological damage at 0–4 h after SCI when the data were compared with those of the uninjured cords (Fig. 2T). The severity of histologically visible overall injury increased significantly in the 1–3-day survival group, in comparison with the control and 0–4 h survival interval groups and then remained constant as survival times increased (Fig. 2T). Although the overall injury scores included areas of necrosis and cavity formation (Zone 1), this score also addressed pathological changes outside these areas (Zone 2). For this reason, and particularly because of the widespread bleeding and oedema in the cords at early time intervals, the overall injury scores were inflated at the early intervals (0–4 h and 1–3 days). As time progressed, the overall injury was more dominated by necrosis and cavity formation, and less by the changes in Zone 2, as haemorrhage and oedema resolved. Alternatively, but less likely, the increase in areas classified as Zone 1 may be spurious because, as cysts form, the areas that once were necrotic become more easily recognized. The changing contributions of Zones 1 and 2 summed to cause a relatively constant overall state of injury, in comparison to the increasing areas of necrosis and cavity formation. With the exception of four cases with ‘laceration’ injuries, all cases were examples of ‘contusion/cyst’ or ‘massive compression’ injuries (Table 1). The necrosis/cavity formation and severity of SCI in the laceration cases resembled that of the compression injuries with the exception of the pial breaches, and, therefore, these cases were included in both analyses. Neutrophils enter the lesion site within hours to a few days after injury Neutrophils in blood vessels and in relatively intact tissue within the injured spinal cord had intracellular immunoreactivity for defensin. These cells also had striking extracellular halos of immunoreactive product, because defensin is a secreted protein (Schluesener and Meyermann, 1995; Barnathan et al., 1997). In the control cases and in uninjured cord segments of SCI, defensin-immunoreactive (-ir) neutrophils were all intravascular and had extracellular halos of defensin immunoproduct (Fig. 3A). At the earliest time interval after SCI (0–4 h), neutrophils were endovascular or were found in petechial haemorrhages in the injured tissue (Fig. 3B). Extravasation of neutrophils from small blood vessels was first observed at 3–4 h after injury. This change in location of the cells (i.e. in haemorrhages or in the process of migrating, or having migrated, through vessel walls) was not yet associated with an increase in ‘free’ extravascular neutrophils in the spinal cord parenchyma. At 1–3 days after SCI, neutrophil infiltration was striking and diffuse throughout grey and white matter, and numerous neutrophils were located in and around central grey haemorrhagic, necrotic areas (Fig. 3C, D and G). Neutrophils in these regions at 5–10 days after injury were smaller than the extravascular neutrophils at 1–3 days after SCI (Fig. 3E versus C), and their lobular nuclei were condensed, suggesting apoptosis. These nuclear changes rendered these cells much more difficult to identify in H&E-stained sections. At 1–5 days after SCI, in areas of necrosis and haemorrhage, the neutrophils had markedly reduced or absent extracellular halos of defensin (Fig. 3D and E), perhaps due to depletion of this secreted protein. Weeks to months after SCI, only a few extravascular neutrophils were scattered through the necrotic areas (Fig. 3F). As the injury-to-death interval lengthened, neutrophils were less likely to be within the tissue parenchyma and more likely to be within blood vessel lumina. Of the eight cases in the weeks-to-months category, only those with the longest survival (the case at 7 months and the case at 1 year) had neutrophils exclusively in blood vessel lumina, resembling the distribution of these cells in control cases (Fig. 3A). Fig. 3 Open in new tabDownload slide Time course of neutrophil infiltration in SCI visualized by defensin immunohistochemistry. (A) Neutrophils in control cases have an extracellular halo of immunoreactivity (arrowhead) and are restricted to the lumen of the blood vessel. (B) At 3 h after SCI, neutrophils accompany red blood cells into the injured tissue at sites of torn tissue (area of haemorrhage indicated by arrowhead). (C) One day after injury numerous neutrophils have migrated through the blood vessel walls (arrows = representative neutrophils; arrowhead = neutrophil diapedesis; bv = blood vessel; inset scale bar: 10 μm). (D) Neutrophils remain prevalent in injured tissue 2 and 3 days after SCI. No pericellular defensin-ir halos are present at this and later stages (arrow, representative neutrophils). (E) The number of neutrophils remains increased above control numbers in areas of tissue disruption 5 days after injury. Note the apparent shrinkage of neutrophils compared with those illustrated in (B) and (C) (arrows indicate representative neutrophils; inset scale bar: 10 μm). (F) Numbers of neutrophils (arrows) in areas of SCI are diminished 3 weeks after injury. Scale bar: 50 μm and applies to all panels except insets. (G) Defensin-stained photomontages of control (no SCI) spinal cord and of lesion centres at 4 h (4 h), 3 days (3 d), 10 days (10 d) and 3 weeks (3 wk) after injury (scale bar: 2.5 mm). (H) The values are the mean percent area of defensin immunoreactivity ± SEM for pooled data from control cases (no SCI, n = 7) and from cases with the following survival intervals after SCI: 0–4 h (n = 8), 1–3 days (n = 5), 5–10 days (n = 6), and weeks to months (n = 7). Asterisks indicate that the 1–3 day group is significantly different from all other groups except the 5–10 day group; 5–10 day group is significantly different from 0–4 h (one-way ANOVA, F4,28 =15.23, P < 0.001, Fisher's protected t-test, P < 0.05). The immunolabelling in the 5–10 day group was marginally greater than that in the control group (one tailed, P = 0.06) Fig. 3 Open in new tabDownload slide Time course of neutrophil infiltration in SCI visualized by defensin immunohistochemistry. (A) Neutrophils in control cases have an extracellular halo of immunoreactivity (arrowhead) and are restricted to the lumen of the blood vessel. (B) At 3 h after SCI, neutrophils accompany red blood cells into the injured tissue at sites of torn tissue (area of haemorrhage indicated by arrowhead). (C) One day after injury numerous neutrophils have migrated through the blood vessel walls (arrows = representative neutrophils; arrowhead = neutrophil diapedesis; bv = blood vessel; inset scale bar: 10 μm). (D) Neutrophils remain prevalent in injured tissue 2 and 3 days after SCI. No pericellular defensin-ir halos are present at this and later stages (arrow, representative neutrophils). (E) The number of neutrophils remains increased above control numbers in areas of tissue disruption 5 days after injury. Note the apparent shrinkage of neutrophils compared with those illustrated in (B) and (C) (arrows indicate representative neutrophils; inset scale bar: 10 μm). (F) Numbers of neutrophils (arrows) in areas of SCI are diminished 3 weeks after injury. Scale bar: 50 μm and applies to all panels except insets. (G) Defensin-stained photomontages of control (no SCI) spinal cord and of lesion centres at 4 h (4 h), 3 days (3 d), 10 days (10 d) and 3 weeks (3 wk) after injury (scale bar: 2.5 mm). (H) The values are the mean percent area of defensin immunoreactivity ± SEM for pooled data from control cases (no SCI, n = 7) and from cases with the following survival intervals after SCI: 0–4 h (n = 8), 1–3 days (n = 5), 5–10 days (n = 6), and weeks to months (n = 7). Asterisks indicate that the 1–3 day group is significantly different from all other groups except the 5–10 day group; 5–10 day group is significantly different from 0–4 h (one-way ANOVA, F4,28 =15.23, P < 0.001, Fisher's protected t-test, P < 0.05). The immunolabelling in the 5–10 day group was marginally greater than that in the control group (one tailed, P = 0.06) Neutrophils were no more numerous in cases of laceration than in cases of contusion or massive compression with the same survival interval. Figure 3G (3 weeks) illustrates a spinal cord with a laceration injury that contains only a few neutrophils. Accordingly, data from these cords were included in the quantitative analysis of neutrophil infiltration. Quantification and time course of neutrophil infiltration in SCI Uninjured spinal cords and cords at 0–4 h after SCI had only a small amount of defensin immunoreactivity (0.060 ± 0.014 and 0.067 ± 0.028%, respectively; Fig. 3G and H). At 1–3 days following injury, a significant 15-fold increase (compared to the control cases and cases with 0–4 h survival) in the area of defensin immunoreactivity occurred (0.88 ± 0.23%). This increase waned to a still significant 3-fold increase at 5–10 days after SCI (0.17 ± 0.040%). The area of defensin immunoreactivity was not different from controls or cases at the 0–4 h interval at weeks to months after SCI (0.10 ± 0.019%). The defensin immunoreactivity (0.093%) in the case at one year after SCI was the same as that in the control cases. At no time was the neutrophil influx significantly different between grey matter and white matter. Activation of microglia and infiltration of monocyte/macrophages at the lesion site within days of SCI In control cases and within 0–4 h of SCI, scant to moderate numbers of CD68-ir microglia were evenly distributed throughout the grey and white matter (Fig. 4A and B). These microglia had particulate cytoplasmic immunolabelling and ramified morphology. The processes of the ramified microglia were more distinct in grey matter than in white matter (Fig. 4A and B), and microglia in white matter tended to be more prominent around blood vessels. In addition, CD68-ir perivascular macrophages were noted (Fig. 4B). At 1–3 days after SCI, microglia entered a transition phase in which their processes had become shorter and thicker and their cell bodies larger, due to activation (Fig. 4C). At this time, the number of activated microglia increased and they were arranged in a patchy distribution around the margins of lesions. However, in areas of preserved white matter, CD68 immunoreactivity appeared to be within ramified microglia, much like that in control spinal cords. At 1–3 days after SCI, small numbers of CD68-ir monocytes, identified by their characteristic nuclear morphology, were found adherent to the endothelium of blood vessels and in adjacent extravascular spaces. These monocytes were likely extravasating into the injury site. A few migrating monocytes were also found in the 5–10 day cases. At 5–10 days (Fig. 4D), in areas of necrosis and lining the inner edges of cavities, CD68-ir cells were numerous and usually rounded with variegated cytoplasmic immunolabelling. Their morphology defined them as phagocytic ‘foamy’ macrophages. Early activated microglia possessing shorter processes and/or rounded cell bodies, were identified in regions adjacent to areas of necrosis (Fig. 4E). However, once the foamy macrophage morphology was established, the microglial or haematogenous origin of the CD68-ir cells could not be determined. In one unique case of central grey necrosis (Fig. 4I: 5d and T8) the entire grey matter was composed of foamy CD68-ir phagocytic macrophages, whereas the white matter contained only ramified microglial with no evidence of such phagocytic macrophages or of microglial activation, and therefore was similar to that of control cords. CD68 immunoreactivity of the phagocytic macrophages varied in intensity: strongly labelled cells tended to be small, with less cytoplasm; weakly labelled cells were large, with foamy cytoplasm. A moderate to marked increase in the number of activated microglia occurred at the margins of SCI and around axonal swellings in grey and white matter. Weeks to 1 year after SCI, the lesion contained many foamy macrophages (Fig. 4F–H). CD68 immunoreactivity, when present, was in these macrophages (Fig. 4F and G) but, at these survival times, most of the cases contained numerous foamy macrophages (in H & E sections) that were not immunoreactive for CD68 (Fig. 4G and H, see inset in G and I ‘2 week C7’). However, in a subpopulation of cases, expression of CD68 could be very great at weeks after SCI as shown in Fig. 4F and I, ‘3 wk C7’). No differences in CD68 immunoreactivity were observed between the laceration, contusion/cyst and massive compression-type injuries at any time interval after SCI and all types were included in the quantitative analysis. Fig. 4 Open in new tabDownload slide Time course of microglia and monocyte/macrophage presence in the injured spinal cord visualized with anti-CD68 antibody. (A and B) CD68 immunoreactivity is present in the processes of ramified microglia in the grey matter in a control case (A: asterisk = motor neuron) and in ramified microglia in white matter in a SCI case 1 h after injury (B: bv = blood vessel). The CD68 immunoreactivity is evenly distributed, particulate and cytoplasmic; labelled cells have discrete, thin processes (arrowheads). (C) By 3 days CD68-ir cells (arrowheads) are more rounded and have shorter processes than those in panels A and B. This morphology is typical of activated microglia. Scale bar (10 μm) on panel C applies to A–C. (D) At 5 days after SCI, large, round CD68-ir cells are in areas of maximal injury (bv = blood vessel). (E) In areas adjacent to injury, the morphology of CD68-ir cells suggests that they are activated microglia (arrowhead). (F) At 3 weeks after SCI CD68-Ir cells are numerous and their cytoplasm is foamy. (G and H) Months to 1 year after injury, cells are rarely CD68-ir (red arrows = macrophages with no CD68; black arrows = CD68-ir macrophages), despite the presence of numerous cells with macrophage morphology in H & E sections (G inset, arrow). Scale bar in H (50 μm) also applies to the inset and to D–G. (I) CD68-stained photomontages of control (no SCI) spinal cord and of lesion centres at 0 h (DAS), 1 day (1 d), 5 days (5 d), 2 weeks (2 wk), and 3 weeks (3 wk) after injury (scale bar: 2.5 mm). The example at 3 weeks contained very large areas of CD68 immunoreactivity, and is a statistical outlier from the remainder of the cases in the weeks-to-months group. This case was not included in the histogram in J. (J) The values are the mean percent areas of CD68 immunoreactivity ± SEM for pooled data from control cases (no SCI, n = 7) and from cases with the following survival intervals after SCI: 0–4 h (n = 8), 1–3 days (n = 5), 5–10 days (n = 6), and weeks to months (n = 5). Asterisks indicates 1–3, 5–10 days and weeks-to-months groups are significantly different from uninjured control; 5–10 days and weeks-to-months groups are significantly different from 0 to 4 h group (one-way ANOVA, F4,26 = 12.24, P < 0.001, Fisher's protected t-test, P < 0.05). Fig. 4 Open in new tabDownload slide Time course of microglia and monocyte/macrophage presence in the injured spinal cord visualized with anti-CD68 antibody. (A and B) CD68 immunoreactivity is present in the processes of ramified microglia in the grey matter in a control case (A: asterisk = motor neuron) and in ramified microglia in white matter in a SCI case 1 h after injury (B: bv = blood vessel). The CD68 immunoreactivity is evenly distributed, particulate and cytoplasmic; labelled cells have discrete, thin processes (arrowheads). (C) By 3 days CD68-ir cells (arrowheads) are more rounded and have shorter processes than those in panels A and B. This morphology is typical of activated microglia. Scale bar (10 μm) on panel C applies to A–C. (D) At 5 days after SCI, large, round CD68-ir cells are in areas of maximal injury (bv = blood vessel). (E) In areas adjacent to injury, the morphology of CD68-ir cells suggests that they are activated microglia (arrowhead). (F) At 3 weeks after SCI CD68-Ir cells are numerous and their cytoplasm is foamy. (G and H) Months to 1 year after injury, cells are rarely CD68-ir (red arrows = macrophages with no CD68; black arrows = CD68-ir macrophages), despite the presence of numerous cells with macrophage morphology in H & E sections (G inset, arrow). Scale bar in H (50 μm) also applies to the inset and to D–G. (I) CD68-stained photomontages of control (no SCI) spinal cord and of lesion centres at 0 h (DAS), 1 day (1 d), 5 days (5 d), 2 weeks (2 wk), and 3 weeks (3 wk) after injury (scale bar: 2.5 mm). The example at 3 weeks contained very large areas of CD68 immunoreactivity, and is a statistical outlier from the remainder of the cases in the weeks-to-months group. This case was not included in the histogram in J. (J) The values are the mean percent areas of CD68 immunoreactivity ± SEM for pooled data from control cases (no SCI, n = 7) and from cases with the following survival intervals after SCI: 0–4 h (n = 8), 1–3 days (n = 5), 5–10 days (n = 6), and weeks to months (n = 5). Asterisks indicates 1–3, 5–10 days and weeks-to-months groups are significantly different from uninjured control; 5–10 days and weeks-to-months groups are significantly different from 0 to 4 h group (one-way ANOVA, F4,26 = 12.24, P < 0.001, Fisher's protected t-test, P < 0.05). Quantitative analysis and time course of microglia/macrophages in the lesion site after SCI The detectable ramified CD68-ir microglia in uninjured cords and at 0–4 h after SCI occupied a very small percentage of the area of the spinal cord (uninjured: 0.048 ± 0.012% and 0–4 h: 0.10 ± 0.043%, Fig. 4I and J). At 1–3 days following injury, the abundance of ramified microglia and the increased size of activated microglia, as they hypertrophied and became more rounded, contributed to the almost 6-fold significant increase in total area of immunoreactivity (0.28 ± 0.17%), compared to that of the control cords. At 5–10 days and weeks to months after injury, the area occupied by CD68-ir microglia/macrophages was still significantly greater (0.33 ± 0.18% and 0.36 ± 0.11%, respectively) than that in controls and in cords at 0–4 h after injury. The area of CD68-ir remained significantly increased in the weeks-months survival time interval, despite reduced expression of CD68 by the foamy macrophages, because of the abundance of these cells in the lesion (Fig. 4G and H). The weeks-to-months group also contained a subpopulation of cases that had very large areas of CD68 immunoreactivity (2.60 and 14.33%) due to intense CD68 expression in almost all of the macrophages. Data from these cases were not included in the histogram of Fig. 4J, as they were statistical outliers. The single case at 1 year after SCI had many foamy macrophages within the lesion centre but very little expression of CD68 (0.009%, Fig. 4H). Lymphocytes infiltrate the injured spinal cord All CD8+ cells were uniform in shape and size and had the characteristic rounded cytological appearance of lymphocytes. In control cases and in cases surviving for 0–4 h and 1–3 days after injury, rare, scattered, solitary CD8+ lymphocytes were located inside blood vessels, in perivascular spaces and in petechial haemorrhages (Fig. 5A and G). Areas of haemorrhage, necrosis and tissue fragmentation, at 1–3 and 5–10 days after injury, contained a few extravascular CD8+ lymphocytes (Fig. 5B and G). As the time after injury increased from weeks to months, CD8+ lymphocytes were encountered more often in some of the cases. In this subset of cases the numbers of CD8+ lymphocytes ranged from a few to groups of 10–20 cells per ×40 field (Fig. 5G). These groups were found in perivascular spaces in regions of tissue damage, including areas at the margins of cystic cavities, and usually were randomly distributed among macrophages (Fig. 5C–E). CD4+ lymphocytes followed the same pattern of distribution as CD8+ lymphocytes but were fewer (compare Fig. 5E and F). We could not detect any CD20+ B lymphocytes in the injured spinal cord. Fig. 5 Open in new tabDownload slide CD8+ and CD4+ cells infiltrate the injured spinal cord weeks after SCI. (A) At 2 h after injury, rare, scattered solitary CD8+ cells (arrowhead) are located inside blood vessels and in the perivascular space (bv = blood vessel). (B) Five days after injury, rare, single CD8+ T-lymphocytes are scattered throughout the cord parenchyma, but they are found mostly within and around blood vessels (arrowhead = extravascular CD8+ cell). (C) Five weeks after injury, groups of 10–20 CD8+ cells are randomly distributed among macrophages and in the perivascular spaces at the cystic cavity margins (arrowhead = CD8+ cell; small arrows; foamy macrophages; inset shows CD8+ cells at high magnification). (D) Higher magnification photomicrograph of the section shown in (C), illustrating CD8+ cells (arrowheads) amongst foamy macrophages (arrows). Inset shows H & E staining of an adjacent section that reveals cell with lymphocytic morphology (arrowhead) amongst macrophages (arrows) (E and F) Photomicrographs of adjacent sections of a lesion at 6 months after injury. CD8+ cells (E, arrowhead) and CD4+ cells (F, arrowhead; inset, high magnification) are distributed among macrophages and adjacent to blood vessels (bv = blood vessel). Scale bar (50 μm) on E also applies to all other panels. Inset scale bars: 10 μm. (G) The average number of CD8+ lymphocytes per ×40 high power field (ordinate) is plotted against a log scale of various survival intervals (abscissa). Fig. 5 Open in new tabDownload slide CD8+ and CD4+ cells infiltrate the injured spinal cord weeks after SCI. (A) At 2 h after injury, rare, scattered solitary CD8+ cells (arrowhead) are located inside blood vessels and in the perivascular space (bv = blood vessel). (B) Five days after injury, rare, single CD8+ T-lymphocytes are scattered throughout the cord parenchyma, but they are found mostly within and around blood vessels (arrowhead = extravascular CD8+ cell). (C) Five weeks after injury, groups of 10–20 CD8+ cells are randomly distributed among macrophages and in the perivascular spaces at the cystic cavity margins (arrowhead = CD8+ cell; small arrows; foamy macrophages; inset shows CD8+ cells at high magnification). (D) Higher magnification photomicrograph of the section shown in (C), illustrating CD8+ cells (arrowheads) amongst foamy macrophages (arrows). Inset shows H & E staining of an adjacent section that reveals cell with lymphocytic morphology (arrowhead) amongst macrophages (arrows) (E and F) Photomicrographs of adjacent sections of a lesion at 6 months after injury. CD8+ cells (E, arrowhead) and CD4+ cells (F, arrowhead; inset, high magnification) are distributed among macrophages and adjacent to blood vessels (bv = blood vessel). Scale bar (50 μm) on E also applies to all other panels. Inset scale bars: 10 μm. (G) The average number of CD8+ lymphocytes per ×40 high power field (ordinate) is plotted against a log scale of various survival intervals (abscissa). Expression of MPO in the lesion centre In uninjured spinal cords, and at all time points assessed after injury, all neutrophils expressed MPO (Fig. 6A and B). Double labelling was not consistently successful in our study and instead, we used two methods to establish the cellular localization of MPO. First, by examining MPO-stained sections at high power (×40), all stained cells had morphology typical of neutrophils (i.e. round cells of 10–12 μm diameter with lobular nuclei; Table 2). Next, areas of MPO immunoreactivity in a subset of eight cases at the 1–3 and 5–10 day intervals were analysed and compared with areas of defensin and CD68 in serial sections. The areas of immunoreactivity for MPO and defensin were identical or very similar in the five cases in which high expression of defensin revealed a neutrophil influx (Table 3). Some ramified microglia (1–2 per ×40 microscopic field) in the uninjured control cords or at 0–4 h after SCI also expressed MPO. At 1–3 and 5–10 days after SCI many ramified and activated microglia (up to 8 per ×40 field), close to areas of haemorrhage and tissue damage, expressed MPO (Table 2). Weeks to months after injury, approximately one-quarter of the phagocytic ‘foamy’ macrophages expressed MPO and the intensity of expression was variable (Fig. 6C and D, Table 2). None of the cells identifiable by cytological criteria as ‘foamy’ macrophages or microglia expressed defensin. The comparison of areas of MPO, defensin and CD68 in three cases at 5–10 days after injury suggests that MPO in the cord could be attributed to both cell types at this time (Table 3). Table 2 Expression of oxidative and proteolytic enzymes in cells in the uninjured spinal cord or the injury epicentre Time . Neutrophils . Macrophages* . MPO (%) gp91phox(%) MMP-9 (%) MPO (%) gp91phox (%) No SCI† 100 0 0 <25 <25 0–4 hours 100 100 100 <25 <25 1–3 days 100 <100 <100 <50 <50 5–10 days 100 <50 <25 <50 <25 Week to months 100s <25 0 <25 <50 Time . Neutrophils . Macrophages* . MPO (%) gp91phox(%) MMP-9 (%) MPO (%) gp91phox (%) No SCI† 100 0 0 <25 <25 0–4 hours 100 100 100 <25 <25 1–3 days 100 <100 <100 <50 <50 5–10 days 100 <50 <25 <50 <25 Week to months 100s <25 0 <25 <50 Data are expressed as an approximate percentage of the cell population expressing the protein using the following criteria: 0 = none; <25% = 1–24%; <50% = 25–49%; <75% = 50–74%; <100% = 75–99%; 100% = all. Note that the neutrophils and macrophages have been identified by their cytological and not immunohistochemical features. *Includes cells with full macrophage morphology and rounded activated microglia with some processes. †Neutrophils are intravascular only (other groups include intra- and extravascular neutrophils). Open in new tab Table 2 Expression of oxidative and proteolytic enzymes in cells in the uninjured spinal cord or the injury epicentre Time . Neutrophils . Macrophages* . MPO (%) gp91phox(%) MMP-9 (%) MPO (%) gp91phox (%) No SCI† 100 0 0 <25 <25 0–4 hours 100 100 100 <25 <25 1–3 days 100 <100 <100 <50 <50 5–10 days 100 <50 <25 <50 <25 Week to months 100s <25 0 <25 <50 Time . Neutrophils . Macrophages* . MPO (%) gp91phox(%) MMP-9 (%) MPO (%) gp91phox (%) No SCI† 100 0 0 <25 <25 0–4 hours 100 100 100 <25 <25 1–3 days 100 <100 <100 <50 <50 5–10 days 100 <50 <25 <50 <25 Week to months 100s <25 0 <25 <50 Data are expressed as an approximate percentage of the cell population expressing the protein using the following criteria: 0 = none; <25% = 1–24%; <50% = 25–49%; <75% = 50–74%; <100% = 75–99%; 100% = all. Note that the neutrophils and macrophages have been identified by their cytological and not immunohistochemical features. *Includes cells with full macrophage morphology and rounded activated microglia with some processes. †Neutrophils are intravascular only (other groups include intra- and extravascular neutrophils). Open in new tab Table 3 Comparison of expression of MPO with that of defensin and CD68 Case number . Survival (days) . % Defensin-Ir . % CD68-Ir . % MPO-Ir . 201 1 1.40 0.95 1.49 119 2 0.77 0.09 0.61 162 3 1.14 0.04 0.81 176 3 1.05 0.01 1.11 175 5 0.33 0.06 0.35 206 5 0.05 0.12 0.31 181 10 0.17 0.18 0.23 66 10 0.09 0.14 0.25 Case number . Survival (days) . % Defensin-Ir . % CD68-Ir . % MPO-Ir . 201 1 1.40 0.95 1.49 119 2 0.77 0.09 0.61 162 3 1.14 0.04 0.81 176 3 1.05 0.01 1.11 175 5 0.33 0.06 0.35 206 5 0.05 0.12 0.31 181 10 0.17 0.18 0.23 66 10 0.09 0.14 0.25 Areas of immunoreactivity are expressed as % of area of interest as defined in Material and methods. These measures were made on serial sections from the listed cases. Open in new tab Table 3 Comparison of expression of MPO with that of defensin and CD68 Case number . Survival (days) . % Defensin-Ir . % CD68-Ir . % MPO-Ir . 201 1 1.40 0.95 1.49 119 2 0.77 0.09 0.61 162 3 1.14 0.04 0.81 176 3 1.05 0.01 1.11 175 5 0.33 0.06 0.35 206 5 0.05 0.12 0.31 181 10 0.17 0.18 0.23 66 10 0.09 0.14 0.25 Case number . Survival (days) . % Defensin-Ir . % CD68-Ir . % MPO-Ir . 201 1 1.40 0.95 1.49 119 2 0.77 0.09 0.61 162 3 1.14 0.04 0.81 176 3 1.05 0.01 1.11 175 5 0.33 0.06 0.35 206 5 0.05 0.12 0.31 181 10 0.17 0.18 0.23 66 10 0.09 0.14 0.25 Areas of immunoreactivity are expressed as % of area of interest as defined in Material and methods. These measures were made on serial sections from the listed cases. Open in new tab Fig. 6 Open in new tabDownload slide Expression of MPO by neutrophils and macrophages. Defensin-ir neutrophils at 3 days after SCI (A) also express MPO as shown in an adjacent section of the same lesion (B). Arrows in A and B point to neutrophils with characteristic lobular nuclei. At 5 days after SCI, CD68-ir phagocytic macrophages (C, arrows) sometimes express MPO as shown in an adjacent section of the same lesion (D). Many cells do not express MPO (black arrow, D) but a minority are MPO-ir (red arrow). Scale bar: (A–D) 50 μm. Fig. 6 Open in new tabDownload slide Expression of MPO by neutrophils and macrophages. Defensin-ir neutrophils at 3 days after SCI (A) also express MPO as shown in an adjacent section of the same lesion (B). Arrows in A and B point to neutrophils with characteristic lobular nuclei. At 5 days after SCI, CD68-ir phagocytic macrophages (C, arrows) sometimes express MPO as shown in an adjacent section of the same lesion (D). Many cells do not express MPO (black arrow, D) but a minority are MPO-ir (red arrow). Scale bar: (A–D) 50 μm. Expression of gp91phox, a marker of oxidative reactivity, in the lesion centre Immunoreactivity for gp91phox was associated with the plasma membrane of intravascular and perivascular neutrophils at 0–4 h after SCI, but neutrophils did not express this protein in control cases (Fig. 7A, Table 2). At 1–3 days following injury, the majority of neutrophils in and around areas of haemorrhage and necrosis were immunoreactive for gp91phox (Fig. 7B, Table 2). Some neutrophils displayed cytoplasmic as well as membranous labelling whereas others exhibited only membranous labelling. At 5–10 days after SCI, less than half of the neutrophils were gp91phox-ir (Fig. 7C, Table 2). By weeks to months, this proportion became even smaller. Throughout the time course after SCI, scattered endothelial and perivascular cells were immunoreactive for gp91phox (Fig. 7C). Fig. 7 Open in new tabDownload slide Expression of gp91phox, a marker of oxidative reactivity, and MMP-9. (A) Microglia in intact tissue within the lesion centre (arrowhead), and intravascular neutrophils (arrow; bv = blood vessel) express gp91phox 1 h after SCI (B) Numerous neutrophils in areas of haemorrhage and necrosis, express gp91phox at 3 days after SCI (red arrowhead). Some neutrophils do not express gp91phox (black arrow). (C) At 5 days after SCI the majority of macrophages do not express gp91phox (black arrow) whereas neutrophils (red arrow), endothelial cells (not indicated) and pericytes (green arrow) are gp91phox-ir. (D) gp91phox-Ir microglia are diffusely distributed around regions of tissue damage, haemorrhage and necrosis at 6 weeks after SCI. Scale bar: (A–D), 50 μm. (E) MMP-9-Ir cells are found in areas of haemorrhage and tissue disruption as early as 1 h after injury. (F) Numerous neutrophils (identified by their characteristic morphology and by their defensin immunoreactivity in adjacent sections) in areas of haemorrhage and necrosis are highly immunoreactive for MMP-9 at 3 days after injury (arrowhead). Some neutrophils do not express MMP-9 (arrow). (G) By 5 days after injury, only a few scattered neutrophils are MMP-9-ir. Scale bar: (E–G) 50 μm. Fig. 7 Open in new tabDownload slide Expression of gp91phox, a marker of oxidative reactivity, and MMP-9. (A) Microglia in intact tissue within the lesion centre (arrowhead), and intravascular neutrophils (arrow; bv = blood vessel) express gp91phox 1 h after SCI (B) Numerous neutrophils in areas of haemorrhage and necrosis, express gp91phox at 3 days after SCI (red arrowhead). Some neutrophils do not express gp91phox (black arrow). (C) At 5 days after SCI the majority of macrophages do not express gp91phox (black arrow) whereas neutrophils (red arrow), endothelial cells (not indicated) and pericytes (green arrow) are gp91phox-ir. (D) gp91phox-Ir microglia are diffusely distributed around regions of tissue damage, haemorrhage and necrosis at 6 weeks after SCI. Scale bar: (A–D), 50 μm. (E) MMP-9-Ir cells are found in areas of haemorrhage and tissue disruption as early as 1 h after injury. (F) Numerous neutrophils (identified by their characteristic morphology and by their defensin immunoreactivity in adjacent sections) in areas of haemorrhage and necrosis are highly immunoreactive for MMP-9 at 3 days after injury (arrowhead). Some neutrophils do not express MMP-9 (arrow). (G) By 5 days after injury, only a few scattered neutrophils are MMP-9-ir. Scale bar: (E–G) 50 μm. In control cords and at all times after injury, ramified microglia near damaged tissue were clearly gp91phox-ir, but the distribution of such labelled cells within grey and white matter, and labelling intensity, varied among cases (Fig. 7A and D, Table 2). At 1–3 and 5–10 days after SCI, some ramified and activated microglia (1–2 per ×40 field) in areas adjacent to tissue damage expressed gp91phox (Table 2) At 5–10 days and weeks to months after injury some phagocytic macrophages (2–4 per ×40 field) were immunoreactive for gp91phox but the majority did not express this enzyme (Fig. 7C) (Table 2). The gp91phox immunoreactivity of macrophages was pronounced at the plasma membrane with lesser amounts in their cytoplasm. MMP expression in the lesion centre At 0–4 h after injury, neutrophils in blood vessels and in perivascular spaces were strongly immunoreactive for MMP-9 (Fig. 7E, Table 2) whereas no expression of this protein was evident in control cases. Almost all neutrophils, identified by their characteristic nuclear morphology, were strongly MMP-9-ir at 1–3 days after SCI (Fig. 7F, Table 2). At 5–10 days after injury, scattered MMP-9-ir neutrophils were present in necrotic areas but overall the proportion of these MMP-9-ir cells had declined (Fig. 7G, Table 2). The few neutrophils, at weeks to months after SCI, did not express MMP-9, like those in control cases (Table 2). In contrast, neither ramified nor activated microglia were MMP-9-ir at any time point assessed after injury. Phagocytic macrophages also did not express MMP-9 (data not shown). Inflammation and histopathological appearance rostral and caudal to the lesion centre The foregoing descriptions were concerned with the appearances of the SCI at the lesion centre. In most cases, tissue samples from segments near the lesion were available, but did not permit a systematic analysis of the progression of inflammation away from the lesion epicentre because, for different cases, the distance of these samples from the lesion centre or from its grossly evident margins was variable. A detailed analysis of changes in tissue adjacent to the injury in three cases is provided in Fig. 8 and its legend. In general, in the first week after injury, intravascular and perivascular neutrophils and activated microglia were observed in region 1–3 segments rostral and caudal to the lesion centre, but still in areas of grossly detectable injury (Fig. 8A and B). However, the intensity of inflammation was less than at the lesion centre. The changes in the prevalence and immunohistochemical appearances of neutrophils in tissue at the margins of the injury site followed the same time course as neutrophils in the depths of the lesion site. In and near damaged areas of these segments, CD68-ir activated microglia were prevalent in all cases later than 0–4 h after SCI. Ramified microglia were present in all groups examined. Foamy macrophages were rarely encountered outside the lesion, although abundant in the lesion centre and margins from 5 days to several months after injury (Fig. 8C). The lymphocyte distribution in grossly intact segments near the injury epicentre was not different from that in control spinal cords. The pattern of expression of MPO, gp91phox and MMP-9 by neutrophils, microglia and macrophages at the margins of the lesion was not different from that in the lesion centre although it was less abundant owing to the small number of inflammatory cells outside the lesion epicentre (Fig. 8B). The appearance of spinal cord sections obtained >8 segments away from the lesion resembled those in the uninjured controls (Fig. 8A). Fig. 8 Open in new tabDownload slide Inflammation and histopathological appearance rostral and caudal to a lesion centre. (A) Three spinal cord sections from a patient who survived for 3 days showing defensin immunostaining at the lesion epicentre (C3 = 3rd cervical segment), in a segment two levels caudal to the lesion epicentre (C5 = 5th cervical segment), and at 19 levels caudal to the lesion epicentre (L2 = 2nd lumbar segment). Scale bar: 0.25 cm. Shown below the sections are higher power (×40) photomicrographs of defensin-immunostained neutrophils (labelled with lowercase Roman numerals i, iii and v) and CD68-immunostained cells (labelled ii, iv and vi) from the areas that are indicated by boxes on each of the spinal cord sections defined above. Each box and its corresponding photomicrograph are labelled with numerals: (i) numerous extravascular neutrophils are present in the lesion epicentre 3 days after injury (arrows = representative neutrophils); (ii) CD68-ir cells (arrows) have ramified microglial morphology or activated microglial and macrophage morphology in the lesion epicentre; (iii) two segments caudal to the lesion epicentre neutrophils are fewer and are obvious inside blood vessels (bv) but are still within the tissue (arrow); (iv) CD68-Ir cells have ramified and activated microglial morphology (arrow); (v and vi) rare neutrophils 19 segments away from the lesion epicentre, (v, arrow) and CD68-Ir cells with morphology of ramified microglia (vi) are present in the tissue, resembling the findings in uninjured spinal cords.. Asterisks in iii–vi label motor neurons in the sections. Scale bar: 50 μm. (B) Spinal section at T2, two segments caudal to a 5-day-old SCI, immunostained for CD68. A central core of tissue, extruded from the lesion epicentre is also present (arrow) at the base of the posterior columns. Shown beside the section are higher power (×40) photomicrographs of defensin, CD68, MPO and gp91phox immunostaining of an area of inflammation in the ventral white matter identified by a box. The cellular characteristics shown in these photomicrographs are similar to those in the extruded area at the base of the posterior columns. (i) Neutrophils stained by defensin are less numerous than usually found in the lesion epicentre at this time (5 days); (ii) CD68-ir cells have morphology typical of activated microglia and macrophages, but are less numerous than in the typical lesion epicentre; (iii) MPO staining is less intense than at the lesion epicentre and appears in neutrophils (black arrow) and macrophages (red arrow); (iv) gp91phox staining is present in some but not all of the neutrophils (arrows). Scale bar: 50 μm. (C) Longitudinal section of an H & E stained spinal cord section, spanning a 5-week-old lesion extending from C5-7. Scale bar: 0.5 cm. Areas identified by boxes in the whole mount section i–vi are shown at higher power (×40) in the photomicrographs below. (i) Areas of grey matter at a short distance from the rostral margin of this contusion injury contain motor neurons (asterisks) with normal Nissl substance (although they show artefactual hyperchromasia); (ii) the grey matter rostral, and immediately adjacent, to the detectable area of injury contains macrophages (arrow) and lymphocytes [inset shows CD8+ cells (arrowheads) amongst macrophages (arrow)] (bv = normal capillary), (iii) the grey matter at the lesion epicentre is necrotic and pale, and intact cells cannot be seen; (iv) the white matter at the grossly visible caudal margin of the lesion is fragmented and contains foamy macrophages (arrow); (v) areas of grey matter at the caudal edge of the lesion contain abundant foamy macrophages (arrows); (vi) the white matter a short distance caudal to the lesion is essentially normal although rare macrophages are visible (arrows), which may be associated with Wallerian degeneration as opposed to the inflammatory response to the SCI. Scale bar: 50 μm. Fig. 8 Open in new tabDownload slide Inflammation and histopathological appearance rostral and caudal to a lesion centre. (A) Three spinal cord sections from a patient who survived for 3 days showing defensin immunostaining at the lesion epicentre (C3 = 3rd cervical segment), in a segment two levels caudal to the lesion epicentre (C5 = 5th cervical segment), and at 19 levels caudal to the lesion epicentre (L2 = 2nd lumbar segment). Scale bar: 0.25 cm. Shown below the sections are higher power (×40) photomicrographs of defensin-immunostained neutrophils (labelled with lowercase Roman numerals i, iii and v) and CD68-immunostained cells (labelled ii, iv and vi) from the areas that are indicated by boxes on each of the spinal cord sections defined above. Each box and its corresponding photomicrograph are labelled with numerals: (i) numerous extravascular neutrophils are present in the lesion epicentre 3 days after injury (arrows = representative neutrophils); (ii) CD68-ir cells (arrows) have ramified microglial morphology or activated microglial and macrophage morphology in the lesion epicentre; (iii) two segments caudal to the lesion epicentre neutrophils are fewer and are obvious inside blood vessels (bv) but are still within the tissue (arrow); (iv) CD68-Ir cells have ramified and activated microglial morphology (arrow); (v and vi) rare neutrophils 19 segments away from the lesion epicentre, (v, arrow) and CD68-Ir cells with morphology of ramified microglia (vi) are present in the tissue, resembling the findings in uninjured spinal cords.. Asterisks in iii–vi label motor neurons in the sections. Scale bar: 50 μm. (B) Spinal section at T2, two segments caudal to a 5-day-old SCI, immunostained for CD68. A central core of tissue, extruded from the lesion epicentre is also present (arrow) at the base of the posterior columns. Shown beside the section are higher power (×40) photomicrographs of defensin, CD68, MPO and gp91phox immunostaining of an area of inflammation in the ventral white matter identified by a box. The cellular characteristics shown in these photomicrographs are similar to those in the extruded area at the base of the posterior columns. (i) Neutrophils stained by defensin are less numerous than usually found in the lesion epicentre at this time (5 days); (ii) CD68-ir cells have morphology typical of activated microglia and macrophages, but are less numerous than in the typical lesion epicentre; (iii) MPO staining is less intense than at the lesion epicentre and appears in neutrophils (black arrow) and macrophages (red arrow); (iv) gp91phox staining is present in some but not all of the neutrophils (arrows). Scale bar: 50 μm. (C) Longitudinal section of an H & E stained spinal cord section, spanning a 5-week-old lesion extending from C5-7. Scale bar: 0.5 cm. Areas identified by boxes in the whole mount section i–vi are shown at higher power (×40) in the photomicrographs below. (i) Areas of grey matter at a short distance from the rostral margin of this contusion injury contain motor neurons (asterisks) with normal Nissl substance (although they show artefactual hyperchromasia); (ii) the grey matter rostral, and immediately adjacent, to the detectable area of injury contains macrophages (arrow) and lymphocytes [inset shows CD8+ cells (arrowheads) amongst macrophages (arrow)] (bv = normal capillary), (iii) the grey matter at the lesion epicentre is necrotic and pale, and intact cells cannot be seen; (iv) the white matter at the grossly visible caudal margin of the lesion is fragmented and contains foamy macrophages (arrow); (v) areas of grey matter at the caudal edge of the lesion contain abundant foamy macrophages (arrows); (vi) the white matter a short distance caudal to the lesion is essentially normal although rare macrophages are visible (arrows), which may be associated with Wallerian degeneration as opposed to the inflammatory response to the SCI. Scale bar: 50 μm. In some compression-type SCI, pressure at the site of the SCI forces tissue to extrude above (and, less commonly, below) the injured area in a telescope-like fashion such that tissue damage ends in an adjacent segment, most commonly in the base of the posterior columns, in a small circular area (Ito et al., 1997). These circular areas of necrosis were encountered in two cases (3 and 5 days post-injury), one of which is illustrated in Fig. 8B. Inflammatory cells in this tissue were neutrophils, activated microglia and macrophages, expressing defensin, CD68, MPO and gp91phox, like those in the ventral white matter area of the same section (Fig. 8B). Highly ramified and activated microglia were located outside and along the rim of the necrotic area (data not shown). As the inflammation waned with distance from the injury epicentre, the pathology in the spinal cords also diminished (Fig. 8C). Tissue at the margins of the lesion was fragmented and contains foamy macrophages and lymphocytes whereas, at short distances from the lesion margins, tissue was essentially normal with only occasional macrophages. Discussion Our study has demonstrated that the first cells to participate in the inflammatory response to human SCI, namely neutrophils and microglia, can generate a variety of oxidative and proteolytic enzymes that have the capacity to cause secondary injury by enlarging the lesion and potentially worsening neurological dysfunction. Numerous macrophages are present in the injured cord and appear to be actively phagocytic for weeks to months after the injury. Although they can express the oxidative enzymes, they only do so weakly if at all. The early inflammatory response to SCI in humans is an important target for therapeutic intervention. In the minutes to hours after traumatic SCI, ischaemia, oxidative damage, oedema and glutamate excitotoxicity all contribute to substantial secondary damage (Blight, 1985; Tator and Fehlings, 1991; Young, 1993; Kwon et al., 2004). The cellular inflammatory events that follow immediately then may play a major role in expansion of the lesion size. Indeed, although we found a correlation between the areas of necrosis and presence of inflammatory cells, these cells often extended into more intact tissue adjacent to the lesion centre. Their impact on this spared tissue is uncertain. The increase in Zone 1 observed in our study may suggest progressive injury of adjacent Zone 2 regions, perhaps related to inflammation, that may even account for some of the ‘secondary’ effects of SCI, especially worsening spasticity and pain. The first haematogenous inflammatory cell to arrive at a site of injury is the neutrophil. In our study, extravascular neutrophils were in areas of haemorrhage as early as 4 h after injury, likely having entered with the blood. The prevalence of neutrophils reached a maximum 1–3 days after injury and remained significantly increased for up to 10 days. Given the short life span of neutrophils (∼6 h) (Whyte et al., 1993) and the fact that these cells do not proliferate in their target sites, this time course suggests that neutrophil entry into the cord ceases by 10 days after injury. Previously, the neutrophil response after human SCI may have been underestimated (Norenberg et al., 2004; Yang et al., 2004), because they are much less apparent in human tissue stained only by H & E than after immunostaining for defensin or MPO. Extravascular neutrophils were generally found in areas of haemorrhage, necrosis and tissue fragmentation. The prevalence of neutrophils after SCI is associated with the time (1–3 days) when injury to the spinal cord increases abruptly and these cells may initiate the secondary changes causing the later progression of necrosis with time after injury. Animal studies have shown that neutrophils are likely to contribute to tissue damage by releasing a myriad of pro-inflammatory mediators (Taoka et al., 1997, 1998). Several studies in animal models of SCI have shown that blockade of neutrophil influx limits secondary damage after SCI, i.e. is ‘neuroprotective’ (Taoka et al., 1997; Farooque et al., 1999; Chatzipanteli et al., 2000; Gris et al., 2004). Microglia are also an important component of the inflammatory process in response to SCI. Activated microglia were in the lesion by 1 day after SCI, leading to increased areas of CD68 immunoreactivity at the 1–3 day interval after SCI. These areas of activated microglia also correlated with increased tissue damage in the cord, as did the presence of neutrophils. At and beyond the 5–10 day interval after SCI, the predominant inflammatory cells were activated microglia and macrophages. Phagocytic macrophages were mostly located in areas of necrosis and along the edge of cystic cavities whereas activated microglia were outside areas of necrosis and in tissue at the margins of the grossly detectable lesion. The presence of activated microglia, with morphology in transition from resting microglia toward the macrophage phenotype, is consistent with the idea that microglia contribute to the population of phagocytic macrophages in the lesion. We found few extravasating monocyte/macrophages, but the observation in animal studies of a muted macrophage response when monocyte/macrophages were depleted or prevented from entering the injured spinal cord suggests that these cells are part of the phagocytic population (Popovich et al., 1999; Saville et al., 2004). The detection of monocytes at the moment of diapedesis through the small blood vessels at the edges of the injury is very difficult and therefore we cannot draw firm conclusions about the contribution of this process to the macrophage population in human SCI. At months to a year after SCI, most of the foamy macrophages were not immunoreactive for CD68. Expression of CD68 is associated with the phagocytic phenotype of macrophages (Schmitt et al., 2000). Diminished expression of CD68 may occur because the macrophages that have been in the lesion for weeks or months are no longer phagocytic and have stopped producing this lysosomal protein. Once activated, macrophages live for ∼4 weeks but may not be phagocytic throughout their entire lifespan (Ross and Auger, 2002). Alternatively, newly arrived macrophages in the chronic lesion may not express CD68 because the cues in the local environment do not prompt its expression or trigger phagocytosis. This diminished expression of CD68 led to an underestimation of the actual numbers of macrophages within the lesions at the chronic survival intervals. In our study a subpopulation of the cases, in the weeks to months after SCI group, had intense CD68 expression in most of the macrophages. The difference in this subpopulation may reflect genetic variation or factors activating these cells such as inflammatory responses outside the spinal cord. When undergoing an oxidative burst, neutrophils and macrophages in the injured spinal cord generate reactive oxygen species that can cause substantial secondary damage by mediating lipid peroxidation and protein nitration, and by activating redox-sensitive signalling cascades and consumption of nitric oxide (Bao et al., 2004). The enzyme NADPH oxidase is the primary source of reactive oxygen species in phagocytic cells (Brandes and Kreuzer, 2005). We used surface expression of its catalytic subunit gp91phox as a marker in our study (Kim et al., 2005). MPO is also a highly expressed enzyme that contributes to oxidative cell damage (Taoka et al., 1997; Bao et al., 2004). MPO and gp91phox were expressed by neutrophils, activated microglia and macrophages in the injured human spinal cords and maximum expression occurred at 1–3 days after injury, coinciding with the time when neutrophil infiltration is most conspicuous and when microglia are undergoing activation. Neutrophils were usually within necrotic areas, which are also surrounded by activated microglia. The high expression of MPO and gp91phox by neutrophils renders them capable of contributing to secondary damage to the surrounding tissue. Microglia are in close proximity to axons, and may significantly contribute to lipid peroxidation of myelin sheaths. Indeed lipid peroxidation and protein tyrosine nitration have been shown in injured human and rat spinal cord in the subacute period after injury (Bao et al., 2004; Chang, 2006). Our results suggest that neutrophils and activated microglia are the major sources of NADPH oxidase-derived reactive oxygen species in the injured human spinal cord. The majority of foamy macrophages at time points later than 5–10 days after injury did not express MPO or gp91phox. Macrophages filled with tissue debris may no longer generate reactive oxygen species in support of phagocytosis, but yet remain in the cord while debris is degraded by these cells. The role that these macrophages play in chronic tissue injury or wound healing and repair is unclear. This topic has been debated in the general context of neural repair, and although most arguments favoured a destructive role for the macrophage early after injury, some supported the idea that macrophages could secrete beneficial growth factors (Crutcher et al., 2006). Certainly phagocytic removal of degenerating myelin that can inhibit axonal growth must be viewed as essential and beneficial. Likewise phagocytosis of lipid membranes of degenerating cells, in the early phase of injury, would limit lipid peroxidation that itself fosters production of free radicals (Bao et al., 2004). Several studies have demonstrated that macrophages are capable of producing numerous factors such as brain-derived neurotrophic factor and the macrophage-derived protein oncomodulin that appear to promote CNS axon growth and regeneration (Dougherty et al., 2000; Yin et al., 2003, 2006). However, the capacity of macrophages to produce neurotoxic proteins is also clear (Yin et al., 2003). In addition to their distribution in necrotic tissue, macrophages were located in preserved or partially damaged tissue (Zone 2). The presence of macrophages in Zone 2 may foster neurological recovery or, alternatively, contribute to the expanding size of the areas of necrosis and cystic cavity that we observed. The limited availability and technical difficulties of working with archived injured human spinal cord tissue prohibited us from searching further for the beneficial or detrimental factors produced by the macrophages. The impact of lymphocytes on secondary degenerative processes and/or wound healing and repair after SCI remains in question (Hauben et al., 2000; Jones et al., 2004; Crutcher et al., 2006). Most studies suggesting a beneficial effect of T-lymphocytes entail artificially increasing the numbers of those cells by vaccination. Important questions are how many endogenous lymphocytes enter the spinal cord and what do they do there? A study suggesting a negative role for endogenous T-lymphocytes showed that, when their entry to the injured spinal cord is limited by neutralizing the chemoattractant CXCL10, tissue preservation and functional recovery is significantly enhanced (Gonzalez et al., 2003). We found few CD8-expressing cells with lymphocyte-like morphology within 5–10 days after injury. A subset of cases at weeks to months after injury had greater numbers of CD8+. Whereas others have reported no evidence of lymphocyte invasion in injured human cord (Schmitt et al., 2000), CD8+ lymphocytes were scattered among macrophages in the spinal cords that we examined. A recent study of human injured spinal cord also reported few lymphocytes and many macrophages in the lesion epicentre at 15, 20 and 60 days post-injury (Chang, 2006). In a few cases, at weeks to months after injury, we identified a small number of CD4+ cells that appeared to be lymphocytes. The antibody that we used readily labelled CD4+ lymphocytes in our control human appendix sections, but the binding of this antibody to cells in archived spinal cord tissue, may have been hindered simply because of the prolonged storage of formalin-fixed tissues in paraffin wax. In this tissue, the time from death of the tissue to fixation and the period of fixation before immunoprocessing are typically much longer than for surgical samples such as the control appendix. This limitation may have led to an underestimation of the prevalence of the CD4+ lymphocytes and possibly of microglia because of artefactual loss of CD4 binding sites. Previous animal experiments have demonstrated improved blood–CNS barrier integrity, reduced neutrophil recruitment, and improved locomotor recovery after SCI in MMP-9 null mice (Noble et al., 2002). In the present study we also examined MMP-9 expression after SCI because MMP-9 specifically degrades components of the basal lamina of the blood–CNS barrier including gelatin, collagens, elastin and vitronectin, and it has been directly implicated in abnormal vascular permeability and tissue damage (Mun-Bryce and Rosenberg, 1998). MMP-9 also cleaves myelin basic protein and contributes to demyelination of healthy axons that survive the original injury (Noble et al., 2002). All these events destroy tissue and encourage inflammation, and they delay or prevent the creation of a pro-regenerative environment in the spinal cord. Our results suggest that neutrophils are the only leucocytes to generate MMP-9 after SCI in humans, and that they are likely to contribute to early secondary tissue damage and haemorrhagic injury. At weeks to months after SCI, MMP-9 could be involved in remodelling extracellular matrix molecules such as collagen and chondroitin sulphate proteoglycans that form scar tissue and prevent neurite outgrowth (Duchossoy et al., 2001), The absence of neutrophils and the lack of MMP-9 in the macrophages may be disadvantageous to tissue repair in this more chronic period. The human spinal cords analysed in this study are from ‘typical’ cases of SCI with respect to their age, gender and cause of injury (University of Alabama National SCI Statistical Center and the Canadian Paraplegic Association websites). Several factors associated with the use of human tissue may have confounded our analysis of the inflammatory response. For example, we assumed that the length of survival was not dependent on the severity of injury. Other factors that dictate injury severity were largely unknown, including: the magnitude of the physical force causing the injury, the extent of multi-system trauma, genetic influences, lifestyle, undetected pre-existing diseases, drugs taken prior to the trauma and infections or other diseases that occurred after the trauma. Cardiovascular and thermoregulatory status, blood transfusions and subsequent surgical operations may also have affected the spinal cord pathology or inflammation. Although one case had been treated with methylprednisolone, a drug that may have decreased the inflammatory response, this case was retained in our study as it did not differ overtly from other cases in the same time group. In contrast, to prevent an overestimation of the inflammatory response, we excluded all cases of sepsis even though sepsis is a common complication of SCI. The general pathology of human SCI is not greatly different from that of experimental animals, although important specific differences exist. Like contusion injury in humans, the rat contusion injury leads to the formation of microscopic, small and large cavities and fluid-filled cysts (Sroga et al., 2003; Norenberg et al., 2004; Shkrum and Ramsay, 2006). A similar contusion injury in the mouse results in the development of a dense connective tissue matrix, that more closely resembles the long-term effects of some laceration and massive compression injuries in humans (Sroga et al., 2003; Norenberg et al., 2004; Shkrum and Ramsay, 2006). Differences in the inflammatory response may be responsible for the generation of these distinct lesion pathologies. The infiltration of neutrophils into the injury site begins within hours of the injury in humans (as observed in this study), rats and mice (Taoka et al., 1997; Kigerl et al., 2006). The time of maximal neutrophil infiltration in rats (1 day) is shorter than the time we observed in humans. In contrast, neutrophils in mice are present in large numbers for 1–3 days after injury, then disappear until 14 days, when they return in greatly reduced numbers and persist for at least 6 weeks after injury (Kigerl et al., 2006). Therefore, in the context of neutrophil-mediated damage, the rat is a closer model to the human. Microglial and macrophage reactions are similar among humans, rats and mice, with microglial activation beginning immediately after injury and peak numbers of macrophages present 5–10 days after injury (Popovich et al., 1997; Sroga et al., 2003). The macrophage response is sustained for weeks to months in both humans and rodents. Lymphocyte infiltration occurs to a much lesser extent in humans and rats, in comparison to mice. Lymphocyte infiltration in rats is highest between 3 and 7 days post-injury whereas, in mice, significant lymphocyte infiltration is delayed until 14 days after injury (Popovich et al., 1997; Sroga et al., 2003). The increase in lymphocyte infiltration that we observed in a few cases appeared only weeks after injury, like the mouse. Unique to the mouse lesion site are fibroblast-like cells that are likely to be involved in the formation of fibrotic tissue matrix (Sroga et al., 2003). The infiltration of these cells coupled with the larger T-cell response in mice may be responsible for minimizing the development of cysts in mice (Sroga et al., 2003). In general, genetic variability causes strain and species differences in the rodent inflammatory response to SCI. Genetic differences among humans may, as well, impact on their inflammatory response to SCI. Indeed, we observed some heterogeneity in the macrophage and lymphocyte responses of the cases that we studied. In conclusion, cellular inflammatory events after human SCI are similar to those found in experimental investigations in rodents. Although one study in humans (Yang et al., 2004) characterized the cellular localization of pro-inflammatory cytokines in the acute period after SCI and another examined macrophage and lymphocyte distributions in three subacute clinical cases (Chang, 2006), our study has provided a more thorough account of the acute and chronic time course of the cellular inflammatory response after human SCI. The data support the idea that neutrophil entry should be limited and also show that the critical time to limit this entry is within the first 3 days, ideally commencing within 12 h of injury. The similarities between the human and rat SCI during this time suggest that treatments such as the anti-CD11d monoclonal antibody that effectively blocks entry of neutrophils and greatly improves neurological outcome (Gris et al., 2004; Saville et al., 2004), may also be valuable as a clinical neuroprotective therapy. By ∼5 days after SCI, the predominant inflammatory cells were activated microglia and macrophages, which were abundant in the cord for up to a year after injury. Any intervention affecting these cells should be done with concern for the capacity of activated macrophages to produce potentially damaging oxidative and proteolytic enzymes. The phagocytic activity of the macrophages decreases within weeks after the human injury, suggesting that some mild stimulus to promote their phagocytic clearance of tissue debris or secretion of growth factors may be needed. The similarities between the human and rodent inflammatory response after SCI will permit better translation to clinical use of treatments developed in animals. This research was supported by a New Emerging Team Grant from the Canadian Institutes of Health Research (CIHR) and by grants NO1-NS-3-2351 and PO1 NS38665 from the National Institutes of Health of the USA. Ms Fleming held a studentship from the Ontario Neurotrauma Foundation, and currently holds a studentship from CIHR. Dr Weaver was a FORE-SCI (Facility of Research Excellence in SCI) visiting scholar and Ms Fleming held a FORE-SCI studentship at the Miami Project to Cure Paralysis during part of the course of this research. The authors are indebted to Ms Gladys Ruenes at the Miami Project for her superb technical advice. We also appreciate the assistance of colleagues at the Department of Pathology, London Health Sciences Centre: Ms Lynn James who retrieved case materials and Ms Deb Reade and Ms Deb Newman who sectioned some of the spinal cords. The authors also thank Dr Canio Polosa for his constructive suggestions regarding this research. Funding to pay the Open Access publication charges for this article was provided by a New Emerging Team Grant from the CIHR. References Agerberth B , Charo J , Werr J , Olsson B , Idali F , Lindbom L , et al. The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations , Blood , 2000 , vol. 96 (pg. 3086 - 93 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Bao F , Chen Y , Dekaban GA , Weaver LC . Early anti-inflammatory treatment reduces lipid peroxidation and protein nitration after spinal cord injury in rats , J Neurochem , 2004 , vol. 88 (pg. 1335 - 44 ) Google Scholar Crossref Search ADS PubMed WorldCat Bao F , Dekaban GA , Weaver LC . Anti-CD11d antibody treatment reduces free radical formation and cell death in the injured spinal cord of rats , J Neurochem , 2005 , vol. 94 (pg. 1361 - 73 ) Google Scholar Crossref Search ADS PubMed WorldCat Barnathan ES , Raghunath PN , Tomaszewski JE , Ganz T , Cines DB , Higazi A al-R . Immunohistochemical localization of defensin in human coronary vessels , Am J Pathol , 1997 , vol. 150 (pg. 1009 - 20 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Bethea JR . Spinal cord injury-induced inflammation: a dual-edged sword , Prog Brain Res , 2000 , vol. 128 (pg. 33 - 42 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Blight AR . Delayed demyelination, macrophage invasion: a candidate for “secondary” cell damage in spinal cord injury , Cent Nerv Syst Trauma , 1985 , vol. 2 (pg. 299 - 315 ) Google Scholar Crossref Search ADS PubMed WorldCat Brandes RP , Kreuzer J . Vascular NADPH oxidases: molecular mechanisms of activation , Cardiovasc Res , 2005 , vol. 65 (pg. 16 - 27 ) Google Scholar Crossref Search ADS PubMed WorldCat Bunge RP , Puckett WR , Becerra JL , Marcillo A , Quencer RM . Seil FJ . Observation on the pathology of human spinal cord injury. A review and classification of 22 new cases with details from a case of chronic cord compression with extensive focal demyelination , Advances in Neurology , 1993 New York Raven Press Ltd. Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Carlson SL , Parrish ME , Springer JE , Doty K , Dossett L . Acute inflammatory response in spinal cord following impact injury , Exp Neurol , 1998 , vol. 151 (pg. 77 - 81 ) Google Scholar Crossref Search ADS PubMed WorldCat Chang HT . Subacute human spinal cord contusion: few lymphocytes and many macrophages , Spinal Cord , 2006 Advanced online publication, 28 February 2006; doi:10.1038/si.sc.3101910 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Chatzipanteli K , Yanagawa Y , Marcillo AE , Kraydieh S , Yezierski RP , Dietrich WD . Posttraumatic hypothermia reduces polymorphonuclear leukocyte accumulation following spinal cord injury in rats , J Neurotrauma , 2000 , vol. 17 (pg. 321 - 32 ) Google Scholar Crossref Search ADS PubMed WorldCat Crutcher KA , Gendelman HE , Kipnis J , Regino Perez-Polo J , Perry VH , Popovich PG , Weaver LC . Debate: “is increasing neuroinflammation beneficial for neural repair?” , J Neuroimmune Pharm , 2006 1:195--211 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Dougherty KD , Dreyfus CF , Black IB . Brain-derived neurotrophic factor in astrocytes, oligodendrocytes and microglia/macrophages after spinal cord injury , Neurobiol Dis , 2000 , vol. 7 (pg. 574 - 85 ) Google Scholar Crossref Search ADS PubMed WorldCat Duchossoy Y , Horvat JC , Stettler O . MMP-related gelatinase activity is strongly induced in scar tissue of injured adult spinal cord, forms pathways for ingrowing neurites , Mol Cell Neurosci , 2001 , vol. 17 (pg. 945 - 56 ) Google Scholar Crossref Search ADS PubMed WorldCat Farooque M , Isaksson J , Olsson Y . Improved recovery after spinal cord trauma in ICAM-1 and P-selectin knockout mice , Neuroreport , 1999 , vol. 10 (pg. 131 - 4 ) Google Scholar Crossref Search ADS PubMed WorldCat Friese MA , Fugger L . Autoreactive CD8+ T cells in multiple sclerosis: a new target for therapy? , Brain , 2005 , vol. 128 (pg. 1747 - 63 ) Google Scholar Crossref Search ADS PubMed WorldCat Gonzalez R , Glaser J , Liu MT , Lane TE , Keirstead HS . Reducing inflammation decreases secondary degeneration and functional deficit after spinal cord injury , Exp Neurol , 2003 , vol. 184 (pg. 456 - 63 ) Google Scholar Crossref Search ADS PubMed WorldCat Greaves DR , Quinn CM , Seldin MF , Gordon S . Functional comparison of the murine macrosialin and human CD68 promoters in macrophage and nonmacrophage cell lines , Genomics , 1998 , vol. 54 (pg. 165 - 8 ) Google Scholar Crossref Search ADS PubMed WorldCat Gris D , Marsh DR , Oatway MA , Chen Y , Hamilton EF , Dekaban GA , et al. Transient blockade of the CD11d/CD18 integrin reduces secondary damage after spinal cord injury, improving sensory, autonomic, and motor function , J Neurosci , 2004 , vol. 24 (pg. 4043 - 51 ) Google Scholar Crossref Search ADS PubMed WorldCat Hall ED . The neuroprotective pharmacology of methylprednisolone , J Neurology , 1992 , vol. 76 (pg. 13 - 22 ) Google Scholar OpenURL Placeholder Text WorldCat Hauben E , Schwartz M . Therapeutic vaccination for spinal cord injury: helping the body to cure itself , Trends Pharmacol Sci , 2003 , vol. 24 (pg. 7 - 12 ) Google Scholar Crossref Search ADS PubMed WorldCat Hauben E , Butovsky O , Nevo U , Yoles E , Moalem G , Agranov E , et al. Passive or active immunization with myelin basic protein promotes recovery from spinal cord contusion , J Neurosci , 2000 , vol. 20 (pg. 6421 - 30 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Hausmann ON . Post-traumatic inflammation following spinal cord injury , Spinal Cord , 2003 , vol. 41 (pg. 369 - 78 ) Google Scholar Crossref Search ADS PubMed WorldCat Ito T , Oyanagi K , Wakabayashi K , Ikuta F . Traumatic spinal cord injury: a neuropathological study on the longitudinal spreading of the lesions , Acta Neuropathol (Berl) , 1997 , vol. 93 (pg. 13 - 8 ) Google Scholar Crossref Search ADS WorldCat Jones TB , Ankeny DP , Guan Z , McGaughy VM , Fisher L , Basso DM , et al. Passive or active immunization with myelin basic protein impairs neurological function and exacerbates neuropathology after spinal cord injury in rats , J Neurosci , 2004 , vol. 24 (pg. 3752 - 61 ) Google Scholar Crossref Search ADS PubMed WorldCat Jones TB , Hart RP , Popovich PG . Molecular control of physiological and pathological T-cell recruitment after mouse spinal cord injury , J Neurosci , 2005 , vol. 25 (pg. 6576 - 83 ) Google Scholar Crossref Search ADS PubMed WorldCat Kigerl KA , McGaughy VM , Popovich PG . Comparative analysis of lesion development and intraspinal inflammation in four strains of mice following spinal contusion injury , J Comp Neurol , 2006 , vol. 494 (pg. 578 - 94 ) Google Scholar Crossref Search ADS PubMed WorldCat Kim MJ , Shin KS , Chung YB , Jung KW , Cha CI , Shin DH . Immunohistochemical study of p47Phox and gp91Phox distributions in rat brain , Brain Res , 2005 , vol. 1040 (pg. 178 - 86 ) Google Scholar Crossref Search ADS PubMed WorldCat Kwon BK , Tetzlaff W , Grauer JN , Beiner J , Vaccaro AR . Pathophysiology and pharmacologic treatment of acute spinal cord injury , Spine J , 2004 , vol. 4 (pg. 451 - 64 ) Google Scholar Crossref Search ADS PubMed WorldCat Mun-Bryce S , Rosenberg GA , Gelatinase B . modulates selective opening of the blood-brain barrier during inflammation , Am J Physiol , 1998 , vol. 274 (pg. 1203 - 11 ) Google Scholar OpenURL Placeholder Text WorldCat Noble 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 , vol. 22 (pg. 7526 - 35 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Norenberg MD , Smith J , Marcillo A . The pathology of human spinal cord injury: defining the problems , J Neurotrauma , 2004 , vol. 21 (pg. 429 - 40 ) Google Scholar Crossref Search ADS PubMed WorldCat Popovich PG , Wei P , Stokes BT . Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats , J Comp Neurol , 1997 , vol. 377 (pg. 443 - 64 ) Google Scholar Crossref Search ADS PubMed WorldCat Popovich PG , Guan Z , Wei P , Huitinga I , van RN , Stokes BT . Depletion of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomical repair after experimental spinal cord injury , Exp Neurol , 1999 , vol. 158 (pg. 351 - 65 ) Google Scholar Crossref Search ADS PubMed WorldCat Popovich PG , Guan Z , McGaughy V , Fisher L , Hickey WF , Basso DM . The neuropathological, behavioral consequences of intraspinal microglial/macrophage activation , J Neuropathol Exp Neurol , 2002 , vol. 61 (pg. 623 - 33 ) Google Scholar Crossref Search ADS PubMed WorldCat Rabchevsky AG , Streit WJ . Role of microglia in postinjury repair and regeneration of the CNS , MRDD Res Rev , 1998 , vol. 4 (pg. 187 - 92 ) Google Scholar OpenURL Placeholder Text WorldCat Ross JA , Auger MJ . Burke BLCE . The biology of the macrophage , The macrophage , 2002 New York Oxford University Press (pg. 3 - 72 ) Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Saville LR , Pospisil CH , Mawhinney LA , Bao F , Simedria FC , Peters AA , et al. A monoclonal antibody to CD11d reduces the inflammatory infiltrate into the injured spinal cord: a potential neuroprotective treatment , J Neuroimmunol , 2004 , vol. 156 (pg. 42 - 57 ) Google Scholar Crossref Search ADS PubMed WorldCat Schluesener H , Meyermann R . Neutrophilic defensins penetrate the blood-brain barrier , J Neurosci Res , 1995 , vol. 42 (pg. 718 - 23 ) Google Scholar Crossref Search ADS PubMed WorldCat Schmitt AB , Buss A , Breuer S , Brook GA , Pech K , Martin D , et al. Major histocompatibility complex class II expression by activated microglia caudal to lesions of descending tracts in the human spinal cord is not associated with a T cell response , Acta Neuropathol (Berl) , 2000 , vol. 100 (pg. 528 - 36 ) Google Scholar Crossref Search ADS WorldCat Schnell L , Fearn S , Klassen H , Schwab ME , Perry VH . Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord , Eur J Neurosci , 1999 , vol. 11 (pg. 3648 - 58 ) Google Scholar Crossref Search ADS PubMed WorldCat Schwartz M . Macrophages and microglia in central nervous system injury: are they helpful or harmful? , J Cereb Blood Flow Metab , 2003 , vol. 23 (pg. 385 - 94 ) Google Scholar Crossref Search ADS PubMed WorldCat Shkrum MJ , Ramsay DA . Shkrum MJ , Ramsay DA . Craniocerebral trauma and vertebrospinal trauma , Forensic pathology of trauma: common problems for the pathologist. Clifton, NJ , 2006 Humana Press p. 581--4 Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Snedecor G , Cochran WG . Statistical methods , 1989 Iowa Iowa State University Press Google Scholar Sroga JM , Jones TB , Kigerl KA , McGaughy VM , Popovich PG . Rats and mice exhibit distinct inflammatory reactions after spinal cord injury , J Comp Neurol , 2003 , vol. 462 (pg. 223 - 40 ) Google Scholar Crossref Search ADS PubMed WorldCat Taoka Y , Okajima K , Uchiba M , Murakami K , Kushimoto S , Johno M , et al. Role of neutrophils in spinal cord injury in the rat , Neurosci , 1997 , vol. 79 (pg. 1177 - 82 ) Google Scholar Crossref Search ADS WorldCat Taoka Y , Okajima K , Murakami K , Johno M , Naruo M . Role of neutrophil elastase in compression-induced spinal injury in rats , Brain Res , 1998 , vol. 799 (pg. 264 - 9 ) Google Scholar Crossref Search ADS PubMed WorldCat Tator CH , Fehlings MG . Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms , J Neurosurg , 1991 , vol. 75 (pg. 15 - 26 ) Google Scholar Crossref Search ADS PubMed WorldCat van den Berg TK , Dopp EA , Dijkstra CD . Rat macrophages: membrane glycoproteins in differentiation and function , Immunol Rev , 2001 , vol. 184 (pg. 45 - 57 ) Google Scholar Crossref Search ADS PubMed WorldCat Vaziri ND , Lee YS , Lin CY , Lin VW , Sindhu RK . NAD(P)H oxidase, superoxide dismutase, catalase, glutathione peroxidase and nitric oxide synthase expression in subacute spinal cord injury , Brain Res , 2004 , vol. 995 (pg. 76 - 83 ) Google Scholar Crossref Search ADS PubMed WorldCat Wang X , Jung J , Asahi M , Chwang W , Russo L , Moskowitz MA , et al. Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury , J Neurosci , 2000 , vol. 20 (pg. 7037 - 42 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Whyte MK , Meagher LC , MacDermot J , Haslett C . Impairment of function in aging neutrophils is associated with apoptosis , J Immunol , 1993 , vol. 150 (pg. 5124 - 34 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Yang L , Blumbergs PC , Jones NR , Manavis J , Sarvestani GT , Ghabriel MN . Early expression and cellular localization of proinflammatory cytokines interleukin-1Beta, interleukin-6, and tumor necrosis factor-alpha in human traumatic spinal cord injury , Spine , 2004 , vol. 29 (pg. 966 - 71 ) Google Scholar Crossref Search ADS PubMed WorldCat Yin Y , Cui Q , Li Y , Irwin N , Fischer D , Harvey AR , et al. Macrophage-derived factors stimulate optic nerve regeneration , J Neurosci , 2003 , vol. 23 (pg. 2284 - 93 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat Yin Y , Henzl MT , Lorber B , Nakazawa T , Thomas TT , Jiang F , et al. Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells , Nat Neurosci , 2006 , vol. 9 (pg. 843 - 52 ) Google Scholar Crossref Search ADS PubMed WorldCat Young W . Secondary injury mechanisms in acute spinal cord injury , J Emerg Med , 1993 , vol. 11 (pg. 13 - 22 ) Google Scholar PubMed OpenURL Placeholder Text WorldCat © 2006 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2006 The Author(s)
TI - The cellular inflammatory response in human spinal cords after injury
JF - Brain
DO - 10.1093/brain/awl296
DA - 2006-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-cellular-inflammatory-response-in-human-spinal-cords-after-injury-2ipnn7dIng
SP - 3249
EP - 3269
VL - 129
IS - 12
DP - DeepDyve
ER -