Complement component 3 (C3) expression in the hippocampus after excitotoxic injury: role of C/EBPβ

Adult male Wistar rats (8–12 weeks old) were used throughout the study. C/EBPβ^+/+ and C/EBPβ^–/– mice were generated from heterozygous breeding pairs, kindly provided by C. M. Croniger and R. W. Hanson (Case Western Reserve University, Cleveland, OH) [55]. Genotypes were identified using genomic PCR, with DNA prepared from tail using the REDExtract-N-AmpTM tissue PCR kit (XNAT kit, Sigma, St Louis, MO). Five animals from each experimental group were analyzed. All procedures with animals were specifically approved by the “Ethics Committee for Animal Experimentation” of the Instituto de Investigaciones Biomédicas and carried out in accordance with the European Communities Council, directive 2010/63/EEC and National regulations, normative 53/2013. Special care was taken to minimize pain or discomfort of animals.

Adult male rats, C/EBPβ^+/+ and C/EBPβ^–/– mice, were anesthetized by intraperitoneal injection of ketamine (60 mg/kg) and medetomidine (0.125 mg/kg) and positioned in a stereotaxic apparatus (Kopf Instruments, CA). Kainic acid (KA) (0.25 μg in 2.50 μl PBS) was delivered unilaterally into the left hippocampus. Flow rate (1 μl/min) was kept constant with a motorized syringe pump (BASi), and the needle was kept in place for 2 min post-injection before being slowly withdrawn. Control animals of the same age were injected with vehicle. Animals were then housed individually to recover and sacrificed 72 h after lesioning with KA.

Total rat or mouse hippocampal RNA samples (2 μg) were used for the synthesis of complementary DNA (cDNA) by reverse transcription using the Reverse Transcription System (Promega, Madison, WI, USA) with a pd(N)6 random hexamer. Real-time PCR was performed in an ABI Prism machine using the SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and 300 nM concentrations of specific primer. The primers used for the determination of the concentration of both rat and mouse C3 mRNA were 5′-ACC TTA CCT CGG CAA GTT TCT-3′(forward sequence) and 5′-TTG TAG AGC TGC TGG TCA GG-3′(reverse sequence), which synthesized a fragment of 140 bp. In all runs, melting curves were performed to ensure that only one DNA fragment was amplified. Cycle threshold (dilution 1:10) was around 25. Amplification of the 18S rRNA was used for normalization of cDNA loading in the PCR as previously described [56]. The relative mRNA content was determined with the 2-ΔΔCt method [57].

Animals previously anesthetized were perfused transcardially with 4 % paraformaldehyde, and the brains were removed, postfixed, and processed for immunohistochemistry using the diaminobenzidine (DAB) method or double-immunofluorescence analysis, as previously described [58]. The following primary antibodies were used: rabbit polyclonal anti-C3 (Abcam, Cambridge, UK), mouse monoclonal anti-C/EBPβ (Abcam, Cambridge, UK), rabbit polyclonal anti-NeuN (Merck-Millipore, Darmstadt, Alemania), rabbit polyclonal anti-IL-1β (Abcam), rat monoclonal anti-CD11b (clon OX42, Serotec, Oxford, UK), and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, Dako, Glostrup, Denmark) for immunodetection of astrocytes. For immunofluorescence analysis to detect C3, a secondary Alexa-Fluor488 goat anti-rabbit was used in combination with Texas Red Lycopersicon esculentum (tomato lectin; Vector Labs USA, emission at 546) to label microglial cells or Neurotrace fluorescent Nissl stain (emission at 546; Molecular Probes, Madrid, Spain) to stain neurons. The slides processed with DAB were examined with a Nikon Eclipse 80i (Düsseldorf, Germany) microscope, equipped with a Nikon DS-Fi1 digital camera. For double immunofluorescence, a LSM710 laser scanning spectral confocal microscope (Zeiss) was used. Confocal microscope settings were adjusted to produce the optimum signal-to-noise ratio. Orthogonal image acquisition was performed as previously described [59]. Briefly, sections containing the CA3 region of the hippocampus were used for the analysis. Confocal acquisitions with orthogonal projections show co-localization of C3/neurotrace, C3/GFAP, and C3/tomato lectin, over the extent of the cell in consecutive 0.5- or 2-μm z-stacks.

A differential interference contrast (DIC) microscope was used to identify the morphology of cells. The images were acquired using a Nikon 90i microscope equipped with a Plan Apo 100× Ph3 DM objective and connected to Nis-Elements BR software.

To evaluate neuronal degeneration, Fluoro-Jade B staining was used [60]. Briefly, sections mounted in gelatin-coated slides were immersed in 100 % alcohol, followed by 70 % alcohol and distilled water containing permanganate. Slides were then incubated with 0.001 Fluoro-Jade B dye (Chemicon, Temecula, USA) for 30 min at room temperature. After staining, sections were washed with distilled water and mounted with DePeX (Serva).

Frozen hippocampal sections were mounted in gelatin-coated slides. The TUNEL protocol was performed using the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Indianapolis, USA), following the manufacturer’s instructions. Slides were mounted with Vectashield (Vector Laboratories), and TUNEL staining was visualized and imaged using a Nikon Eclipse 80i (Düsseldorf, Germany) microscope, equipped with a Nikon DS-Fi1 digital camera.

First, we validated our models of excitotoxicity by analyzing the damaging effect of KA in the CA3 subfield, which is the most sensitive area of the hippocampus to this excitotoxin. As expected, we found a dramatic increase in the number of degenerating neurons stained with Fluoro-Jade B (Fig. 1a) in rats and mice injected with KA, which was more noticeable in the pyramidal cells of this area. We also found that this cell death occurs via apoptosis since a significant increase in the number of TUNEL⁺ cells (Fig. 1b) was also observed, which was not detected in C/EBPβ-deficient mice (Fig. 1b).

We next studied the content of C/EBPβ and C3 proteins, by immunohistochemistry analysis, in consecutive slices of the hippocampus of adult rats after KA injury. As shown in Fig. 2a, C/EBPβ and C3 proteins are barely detectable in the hippocampus of control animals. In contrast, after KA injection, a dramatic increase of both proteins was observed, which was most prominent in the CA3 subfield of the hippocampus (Fig. 2a). No expression of either C/EBPβ or C3 was detected in other hippocampal areas. It is known that the injection of KA can cause the rupture of the blood brain barrier (BBB) allowing the entry of plasmatic proteins into the brain. Therefore, we next analyzed whether the C3 protein found in the CA3 region after KA injection comes from outside the central nervous system or is due to a local synthesis in the hippocampus. To this end, we studied the hippocampal amount of C3 mRNA by quantitative PCR. Figure 2b shows that in fact C3 mRNA was also increased after KA injection indicating that the observed increase in C3 protein is caused, at least in part, by an increase in the production of C3 gene in brain cells.

Next we analyzed the cell types responsible for the KA-induced increase in C3 observed in the rat hippocampus. For that purpose, we performed double immunostaining in the CA3 region of the rat hippocampus, the region were the induction of C3 is most prominent. As shown in Fig. 3a, double labeling with a C3-specific antibody and neurotrace, a specific marker for neurons, clearly indicates the presence of C3 protein inside the neurons and also in the surrounding area 72 h after KA injection. Given the massive neuronal cell death that takes place in this region of the hippocampus, these observations suggest that C3 may be localized in those neurons that are already degenerating.

After brain injury, there is an increase in the number of reactive glial cells, both active astrocytes and microglial cells as labeled with a GFAP-specific antibody and tomato lectin, respectively. As expected, after KA injection, we observed an increase of both types of reactive glial cells. The presence of C3 protein was detected in both microglial cells and astrocytes, as shown in Fig. 3b, c, respectively. In addition, double-immunofluorescence analysis shows that the increase in C/EBPβ levels observed in wild type animals takes place in neurons (Fig. 3d).

Interestingly an increase in C3 labeling was clearly observed surrounding the microvasculature of the KA-injected hippocampus (Additional file 1) suggesting an implication of this protein in angiogenesis. Additionally, in this Figure, after double staining with a C3-specific antibody and tomato lectin, we show images of C3 localization near microglial cells suggesting also the opsonization and phagocytosis functions of C3 and microglial cells.

Next, we analyzed whether the coexpression of C3 and C/EBPβ in the same areas of the hippocampus also took place in mice. We find also a dramatic increase in the amount of C/EBPβ and C3 proteins in the hippocampus after KA injection. As shown in Fig. 4a, both proteins are almost undetectable in controls in the diverse regions of the hippocampus analyzed. A clear increase is observed after KA injection; however, and in contrast with rats, we could detect C/EBPβ and C3 proteins in three different regions of the hippocampus, CA1, CA3, and dentate gyrus (DG). Figure 4b shows differential interference contrast (DIC) images of C/EBPβ and C3 to show the whole cell. A combination of DAB (C3) and fluorescence (C/EBPβ) staining shows that the increase of both proteins occurs in the same cells (Fig. 4c).

In order to determine the causative implication of C/EBPβ in the KA-induced expression of the C3 gene, we analyzed the effect of KA injection in the hippocampal expression of C3 gene in C/EBPβ^−/− mice. As can be observed in Fig. 5a, C3 protein levels were clearly increased relative to vehicle-injected controls in the hippocampus 72 h following KA injection of C/EBPβ^+/+ animals. On the contrary, no increase in C3 was observed in the hippocampus of C/EBPβ^−/− mice. These data further support the notion that the transcription factor C/EBPβ is a major regulator of C3 gene expression in the brain [54]. Furthermore, our results show that mice lacking C/EBPβ do not present an increase in C3 mRNA levels after the lesion (Fig. 5b), further indicating that indeed C/EBPβ is essential to induce C3 expression in the hippocampus and also suggesting a local production of C3.

One of the events that takes place in the hippocampus after KA injury is the activation of microglial cells and the liberation of proinflammatory cytokines, which is in part responsible for the neuronal degeneration. Glial activation (as shown by GFAP- and OX42-positive cells) and induction of IL1β (a very potent proinflammatory agent) were clearly observed in the hippocampus of wild type mice 72 h after KA injection (Fig. 6). This strong neuroinflammation process was completely absent in the hippocampus of C/EBPβ^−/− mice.