Reduced neuronal cell death after experimental brain injury in mice lacking a functional alternative pathway of complement activation
© Leinhase et al; licensee BioMed Central Ltd. 2006
Received: 28 January 2006
Accepted: 14 July 2006
Published: 14 July 2006
Neuroprotective strategies for prevention of the neuropathological sequelae of traumatic brain injury (TBI) have largely failed in translation to clinical treatment. Thus, there is a substantial need for further understanding the molecular mechanisms and pathways which lead to secondary neuronal cell death in the injured brain. The intracerebral activation of the complement cascade was shown to mediate inflammation and tissue destruction after TBI. However, the exact pathways of complement activation involved in the induction of posttraumatic neurodegeneration have not yet been assessed. In the present study, we investigated the role of the alternative complement activation pathway in contributing to neuronal cell death, based on a standardized TBI model in mice with targeted deletion of the factor B gene (fB-/-), a "key" component required for activation of the alternative complement pathway.
After experimental TBI in wild-type (fB+/+) mice, there was a massive time-dependent systemic complement activation, as determined by enhanced C5a serum levels for up to 7 days. In contrast, the extent of systemic complement activation was significantly attenuated in fB-/- mice (P < 0.05,fB-/- vs. fB+/+; t = 4 h, 24 h, and 7 days after TBI). TUNEL histochemistry experiments revealed that posttraumatic neuronal cell death was clearly reduced for up to 7 days in the injured brain hemispheres of fB-/- mice, compared to fB+/+ littermates. Furthermore, a strong upregulation of the anti-apoptotic mediator Bcl-2 and downregulation of the pro-apoptotic Fas receptor was detected in brain homogenates of head-injured fB-/- vs. fB+/+ mice by Western blot analysis.
The alternative pathway of complement activation appears to play a more crucial role in the pathophysiology of TBI than previously appreciated. This notion is based on the findings of (a) the significant attenuation of overall complement activation in head-injured fB-/- mice, as determined by a reduction of serum C5a concentrations to constitutive levels in normal mice, and (b) by a dramatic reduction of TUNEL-positive neurons in conjunction with an upregulation of Bcl-2 and downregulation of the Fas receptor in head-injured fB-/- mice, compared to fB+/+ littermates. Pharmacological targeting of the alternative complement pathway during the "time-window of opportunity" after TBI may represent a promising new strategy to be pursued in future studies.
The high incidence of adverse outcomes after traumatic brain injury (TBI) has been attributed in large part to secondary mechanisms of neuronal cell death [1, 2]. These include the induction of neuronal apoptosis and complement-mediated neuronal cell lysis [3–7]. Recent evidence suggests that the intracerebral activation of the complement cascade influences the fate of neurons by other than just the "classical" neuroinflammation-mediated effects [8–10]. For example, neuronal apoptosis can be induced by complement activation products, e.g. by binding of the anaphylatoxin C5a to its receptor (C5aR/CD88) expressed on neurons [11–15]. In addition, complement-mediated neuronal cell lysis can occur through the membrane attack complex (MAC; C5b-9) following inactivation of the physiological cellular protection mechanisms against homologous complement-mediated cell death [16–20]. Insights from recent experimental studies on intracerebral MAC injection underline the important role of the membrane attack pathway of complement in contributing to secondary neurodegeneration [21, 22]. Posttraumatic complement activation and tissue deposition of the MAC were furthermore demonstrated in injured human and rodent brains by immunohistochemistry [18, 23–26]. In addition, we have reported elevated levels of soluble MAC in human cerebrospinal fluid (CSF) after severe head injury .
Up to date, most studies which investigated the role of complement activation in the injured brain have focused on the effects of the complement cascade at a point where all three activation pathways converge, i.e. at the level of C3 or further downstream in the cascade [26, 28–33]. Thus, the role which the individual pathways of complement activation play in the pathophysiology of TBI has not yet been determined. Recent studies established the alternative pathway of complement activation as a "key player" in the pathogenesis of ischemia/reperfusion-mediated inflammatory diseases outside the CNS . For example, complement activation in renal ischemia/reperfusion injury was shown to be mediated almost exclusively by the alternative pathway [34–36]. In clinical studies on TBI patients, we have reported elevated levels of the crucial components required for alternative pathway complement activation, factor B and C3, in the CSF of severely head-injured patients .
Here, we demonstrate for the first time an important role of the alternative complement pathway in contributing to posttraumatic neuronal cell death, based on a standardized TBI model in factor B gene-deficient mice.
Results and discussion
Complement activation is attenuated in brain-injured fB-/- mice
Experimental head injury induced mean anaphylatoxin C5a levels in serum of around 50 ng/ml within 7 days in wild-type mice (Fig. 2). Serum C5a levels described in the literature due to triggers for complement activation other than CNS trauma are in the range of 100 ng/ml, as described for patients with severe sepsis . With regard to central nervous system inflammation, we have previously reported significantly elevated C5a levels in human CSF of patients with bacterial meningitis (mean levels around 75 ng/ml), as compared to patients with aseptic/viral meningitis . C5a levels were not detectable in normal CSF of n = 66 healthy humans with a lower limit of sensitivity of the assay at 1.5 ng/ml . Thus, the serum C5a levels detected in brain-injured C57BL/6 mice in the present study appear to reflect concentrations of clinical relevance, as far as data derived from an experimental mouse model can be extrapolated to human disease.
The lack of factor B leads to reduced neuronal cell death after head injury
Upregulation of Bcl-2 and downregulation of Fas in injured fB-/- brains
With regard to the extrinsic pathway of apoptosis, a marked downregulation in Fas receptor expression was seen within 4 hours to 7 days after TBI in fB-/- mice, compared to fB+/+ animals (Fig. 5). Although these data are not quantitative, the differences in staining intensity of the 26 kDa (Bcl-2) and 48 kDa bands (Fas) appear more intense in the brain-injured knockout mice than in the corresponding wild-type littermates at the above-mentioned time-points (Fig. 5). These findings suggest, but do not prove, an involvement of the alternative pathway of complement activation in regulating neuronal apoptosis after TBI by suppression of Bcl-2 and induction of Fas receptor expression in the injured brain. Both aspects are critical in the regulation of post-injury neuronal apoptosis, as previously determined by other investigators in different model systems [48–50]. An experimental study on a controlled cortical impact brain injury model demonstrated that the cortical lesion volume was significantly reduced in transgenic mice with over-expression of the Bcl-2 gene by 7 days after trauma, compared to wild-type littermates . Thus, Bcl-2 was attributed an important role in the regulation of the mitochondrial (intrinsic) pathway of apoptosis after TBI [4, 53, 55, 56].
We have recently shown that the pharmacological "pan"-inhibition of complement activation at the level of the C3 convertases by Crry-Ig, a murine recombinant chimeric fusion molecule, leads to enhanced intracerebral Bcl-2 gene and protein expression and to increased neuronal survival in the hippocampus of brain-injured mice . Similar findings were described in a model of murine autoimmune cerebritis, where the blocking of complement activation by Crry-Ig resulted in a significant attenuation of neuronal apoptosis .
The data from the present study support the biological significance of the alternative pathway of complement activation in contributing to the neuropathological sequelae of TBI and provide the basis for future pharmacological studies with selective alternative pathway inhibitors, e.g. such as factor B antagonists [34, 57].
In summary, the present data provide first evidence of a major role of the alternative pathway of complement activation in contributing to the overall extent of posttraumatic complement activation (C5a generation) and to secondary neuronal cell death after brain injury (TUNEL, Bcl-2, and Fas data). This is a fairly new and provocative notion, since all previously published studies on experimental complement inhibition in TBI models have focussed on interfering with the complement cascade at the "common junction" level of C3 convertases [26, 28–32] or further downstream in the cascade, e.g. by specific blocking of anaphylatoxin C5a or its receptor . The hitherto underestimation of the pathophysiological role of the alternative complement pathway in the neuropathology of brain injury may be in part due to the historically established predominant role of the classical pathway in various neurological diseases [58, 59]. However, the results from the present study imply that these insights may not necessarily reflect the "true" in vivo significance of the alternative complement pathway in a complex multifactorial neuroinflammatory disease, such as in the setting of TBI . The fact that elevated factor B levels are present in the intrathecal compartment of severely head-injured patients  further supports the concept that the pharmacological targeting of factor B may present a reasonable basis for future investigations.
Factor B-/- mice
The genetic knockout mice deficient in factor B (fB-/-) were previously characterized and shown to have a complete lack of a functional alternative complement pathway . They were originally created with Sv129 embryonic stem cells and crossed with C57BL/6 mice prior to expansion of the colony at F1. They were then back-crossed for more than 10 generations against a pure C57BL/6 background and found to be grossly indistinguishable from C57BL/6 mice . Knockout mice and wild-type littermates (fB+/+) were acclimatized several weeks before the experiments and kept isolated from external influences during the entire time course of the study. They were bred in a selective pathogen-free (SPF) environment and standardized conditions of temperature (21°C), humidity (60%), light and dark cycles (12:12 h), with food and water provided ad libitum. Only male mice were used for this study in order to avoid a bias in gender with regard to levels of complement activity and to susceptibility to brain injury which seems to be significantly influenced by female reproductive hormones [62, 63]. All experiments were performed in compliance with the standards of the Federation of European Laboratory Animal Science Association (FELASA) and were approved by the institutional animal care commitee (Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit, Berlin, Germany, No. G0099/03 and No. G0308/04).
Brain injury model
Experimental closed head injury was performed in knockout (fB-/-) mice and wild-type littermates (fB+/+) of the C57BL/6 strain (n = 6 per group and time-point) using a standardized weight-drop device, as previously described [13, 41, 64–66]. In brief, after induction of isoflurane anesthesia, the skull was exposed by a midline longitudinal scalp incision. A 333 g weight was dropped on the fixed skull from a height of 2 cm, resulting in a focal blunt injury to the left hemisphere. After trauma, the mice received supporting oxygenation with 100% O2 until fully awake and were then brought back to their cages. At defined time-points (t = 4 h, 24 h, and 7 days), mice were euthanized and brain hemispheres were extracted for analysis by immunohistochemistry, TUNEL histochemistry, and SDS-PAGE/Western blot analysis. In addition, serum samples were collected for determination of complement anaphylatoxin C5a levels by ELISA and Western blot analysis of Bcl-2 (see below).
Sham-operated mice were kept under identical conditions as the trauma group and underwent the same procedures (anesthesia and scalp incision) except that no head injury was applied.
ELISA for mouse C5a
For determination of complement anaphylatoxin C5a levels in serum samples of head-injured and normal C57BL/6 control mice, we used an ELISA developed in the laboratory of Dr. P.A. Ward (Ann Arbor, MI, USA), as previously described . In brief, ELISA plates (Immulon 4HBX, Thermo Labsystems, Milford, MA, USA) were coated with purified monoclonal anti-mouse C5a IgG (5 μg/ml, BD Pharmingen, San Diego, CA, USA). This capture antibody recognizes both C5a and C5a-desArg, but not the precursor molecule C5. After blocking of non-specific binding sites with 1% milk (Roth, Karlsruhe, Germany) in PBS (Gibco-Invitrogen, Carlsbad, CA, USA) containing 0.05% TWEEN 20 (Sigma-Aldrich, St. Louis, MO, USA), the plate was coated with 100 μl serum diluted 1:20 (in 0.1% milk in PBS contaning 0.05% TWEEN) and murine recombinant mouse C5a at defined concentrations for establishing the standard curve. After incubation and subsequent washing steps, biotinylated monoclonal anti-mouse C5a antibody was added at 500 ng/ml (BD Pharmingen) followed by washing steps and incubation with streptavidin-peroxidase at 400 ng/ml (Sigma). For colorimetric reaction, the substrate (0.4 mg/ml OPD with 0,4 mg/ml urea hydrogen peroxide in 0.05 M phosphate citrate buffer; Sigma) was added and the color reaction was stopped with 3 M sulfuric acid. The absorbance was read at 490 nm ("SpectraMax 190" reader, Molecular Devices, Sunnyvale, CA, USA. All samples were analyzed in duplicate wells and results were calculated from the means of duplicate sample analysis. The standard curve was linear from 50 ng/ml to 0.1 ng/ml which represents the lower limit of detection of this assay.
All mice used in this study were screened by Western blot analysis for the presence of factor B in serum, as an internal quality control (Fig. 1). The protein levels of the mitochondrial anti-apoptotic mediator Bcl-2 and of the pro-apoptotic Fas receptor were determined in homogenized mouse brains and matched serum samples at 4 h, 24 h and 7d following head injury or sham operation in fB-/- and fB+/+ mice. The Western blot technique was previously described . Briefly, mouse brains were extracted under anesthesia, separated into left and right hemispheres, and immediately homogenized in lysis buffer (Sigma) containing 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 0.5% sodium dodecyl sulfate (SDS), 0.5% Nonidet P-40, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 μg/ml pepstatin, 1 mM phenyl-methyl-sulfonyl fluoride in deionized water, using an Ultra Turrax Homogenizer® (IKA Werke, Staufen, Germany). After 15 min centrifugation at 13,000 × g, the protein content of the supernatants was determined by commercially available colorimetric protein assay ("BCA Protein Assay", Pierce/Perbio Science, Bonn, Germany). A 60 μg sample of total protein was denatured in loading buffer and separated under reducing conditions on 12% SDS-polyacrylamide gels in parallel with a broad range prestained SDS-PAGE protein standard (Bio-Rad, Munich, Germany). Proteins were then transferred to Protran BA 83 nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) by electroblotting (Bio-Rad). Equal transfer of protein to the blotting membrane was confirmed by ponceau red staining (Sigma). The blots were blocked overnight and then incubated with either monoclonal anti-mouse Bcl-2 (Santa Cruz Biotechnology, Heidelberg, Germany), diluted 1:500, polyclonal rabbit anti-mouse Fas (clone A-20, Santa Cruz), diluted 1:200, polyclonal chicken anti-mouse anti-factor B, diluted 1:8,000 (kindly provided by Dr. Scott R. Barnum, University of Alabama at Birmingham, AL, USA) as primary antibodies, and with a monocloncal anti-β-actin antibody (clone AC-15, Sigma) diluted 1:10,000, as internal control for ascertaining equal loading of the bands. After incubation with peroxidase-labelled secondary antibodies (Dako, Hamburg, Germany, and Santa Cruz Biotechnology, Heidelberg, Germany), diluted 1:5,000, antibody binding was visualized by a non-radioactive chemiluminescence technique using a commercially available ECL® Western blotting kit (Amersham Pharmacia Biotech, Freiburg, Germany). A semi-quantitative analysis of the individual band intensities was performed by scanning the films (hp Scanjet 5530, Hewlett-Packard, Böblingen, Germany) followed by quantification using the TINA 2.09 software (Raytest, Straubenhardt, Germany). The data are presented in histograms as relative levels to the according β-actin band intensity.
For assessment of neuronal morphology, integrity, and apoptosis, extracted mouse brains were snap-frozen in liquid nitrogen, embedded in OCT compound (Sakura Finetek, Torrance, CA) and stored at -80°C until used for analysis. Six to eight-micrometer thick coronal tissue sections were cut with a cryostat at -20°C. For immunohistochemistry, slides were fixed in acetone and then analyzed by a standard biotin/avidin/peroxidase technique with DAB-tetrahydrochloride as chromogen (Vector, Burlingame, CA), as previously described [13, 32]. The following primary antibodies were used as cell-markers: monoclonal anti-NeuN, at a titrated dilution of 1:2,000 (Chemicon, Hampshire, UK) for neurons; polyclonal rabbit anti-GFAP, 1:100 (Shandon Immunon, Pittsburgh, PA, USA) for astrocytes; monoclonal rat anti-CD11b, 1:100, (Accurate Chemical, Westbury, NY, USA) for microglia; polyclonal goat-anti CD144, 1:200 (Santa Cruz) for endothelial cells. Non-immunized IgG (Vector) was used as negative control at equal dilutions as the omitted specific antibody.
To determine the extent of intracerebral neuronal cell death, TUNEL histochemistry was performed using a "Fluorescein In Situ Cell Death Detection Kit" (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's instructions, as previously described . Briefly, slides were dried for 30 min followed by fixation in 10% formalin solution at RT. After washing in PBS (three times for 3 min), sections were incubated in ice-cold ethanol-acetic acid solution (2:1) for 5 min at -20°C. Thereafter, they were washed in PBS and incubated in a permeabilization solution with 3% Triton X-100 in PBS for 60 min at RT, then incubated with the TdT enzyme in a reaction buffer containing fluorescein-dUTP for 90 min at 37°C. Negative control was performed using only the reaction buffer without TdT enzyme. Positive controls were performed by digesting equal brain sections with DNase grade I solution (500 U/ml; Roche) for 20 min at RT and always kept separate from the other samples thereafter. After labelling, the sections were washed again in PBS and to visualize the unstained (TUNEL-negative) cells, the sections were covered with Vectashield® mounting medium for fluorescence with DAPI (Vector). All samples were evaluated immediately after staining using an Axioskop 40 fluorescence microscope (Zeiss, Germany) at 460 nm for DAPI and 520 nm for TUNEL fluorescence and analyzed by Alpha digi doc 1201 software (Alpha Innotech, San Leandro, CA, USA).
Statistical analysis was performed using commercially available software (SPSS 9.0 for Windows™). Differences in complement C5a levels in serum of fB-/- and fB+/+ mice were determined by the unpaired Student's t-test. A P-value < 0.05 was considered statistically significant.
- C5a receptor (C5aR:
central nervous system
- Crry-Ig :
complement receptor type 1-related protein y chimeric molecule fused to mouse Ig
enyzme-linked immunosorbent assay
factor B gene-deficient mice (fB-/-) and wild-type littermates
glial fibrillary acidic protein
membrane attack complex
neuron-specific nuclear protein
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
traumatic brain injury
terminal deoxynucleotidyl transferase
terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling
We thank Mrs. Claudia Conrad for excellent technical assistance. Dr. Scott R. Barnum (University of Alabama at Birmingham AL, USA) is acknowledged for providing the anti-mouse factor B antibodies used for screening of the knockout and wild-type mice. This study was supported by grants No. STA 635/1-1, STA 635/1-2, STA 635/2-1, and STA 635/2-2 from the German Research Foundation (DFG) to PFS and OIS; NIH grants R01 AI31105 to VMH and K08 DK64790 to JMT; grants GM 61656 and GM 029507 to PAW. The continuing inspiration of EAP is acknowledged.
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