Complement: a novel factor in basal and ischemia-induced neurogenesis
2006; Springer Nature; Volume: 25; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7601004
ISSN1460-2075
AutoresYalda Rahpeymai, Max Albert Hietala, Ulrika Wilhelmsson, Andrew P. Fotheringham, Ioan Davies, Ann-Katrin Nilsson, Jörg Zwirner, Rick A. Wetsel, Craig Gérard, Milos Pekny, Marcela Pekna,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoArticle23 February 2006free access Complement: a novel factor in basal and ischemia-induced neurogenesis Yalda Rahpeymai Yalda Rahpeymai Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Max Albert Hietala Max Albert Hietala Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Ulrika Wilhelmsson Ulrika Wilhelmsson The Arvid Carlsson Institute for Neuroscience, Institute of Neuroscience and Physiology, Section for Clinical Neuroscience and Rehabilitation, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Andrew Fotheringham Andrew Fotheringham School of Medicine and School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Ioan Davies Ioan Davies School of Medicine and School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Ann-Katrin Nilsson Ann-Katrin Nilsson Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Jörg Zwirner Jörg Zwirner Department of Immunology, Georg-August-University Göttingen, Göttingen, Germany Search for more papers by this author Rick A Wetsel Rick A Wetsel Research Center for Immunology and Autoimmune Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas-Houston, Houston, TX, USA Search for more papers by this author Craig Gerard Craig Gerard Pulmonary Division, Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Milos Pekny Milos Pekny The Arvid Carlsson Institute for Neuroscience, Institute of Neuroscience and Physiology, Section for Clinical Neuroscience and Rehabilitation, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Marcela Pekna Corresponding Author Marcela Pekna Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Yalda Rahpeymai Yalda Rahpeymai Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Max Albert Hietala Max Albert Hietala Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Ulrika Wilhelmsson Ulrika Wilhelmsson The Arvid Carlsson Institute for Neuroscience, Institute of Neuroscience and Physiology, Section for Clinical Neuroscience and Rehabilitation, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Andrew Fotheringham Andrew Fotheringham School of Medicine and School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Ioan Davies Ioan Davies School of Medicine and School of Biological Sciences, University of Manchester, Manchester, UK Search for more papers by this author Ann-Katrin Nilsson Ann-Katrin Nilsson Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Jörg Zwirner Jörg Zwirner Department of Immunology, Georg-August-University Göttingen, Göttingen, Germany Search for more papers by this author Rick A Wetsel Rick A Wetsel Research Center for Immunology and Autoimmune Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas-Houston, Houston, TX, USA Search for more papers by this author Craig Gerard Craig Gerard Pulmonary Division, Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Milos Pekny Milos Pekny The Arvid Carlsson Institute for Neuroscience, Institute of Neuroscience and Physiology, Section for Clinical Neuroscience and Rehabilitation, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Marcela Pekna Corresponding Author Marcela Pekna Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden Search for more papers by this author Author Information Yalda Rahpeymai1, Max Albert Hietala1, Ulrika Wilhelmsson2, Andrew Fotheringham3, Ioan Davies3, Ann-Katrin Nilsson1, Jörg Zwirner4, Rick A Wetsel5, Craig Gerard6, Milos Pekny2 and Marcela Pekna 1 1Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden 2The Arvid Carlsson Institute for Neuroscience, Institute of Neuroscience and Physiology, Section for Clinical Neuroscience and Rehabilitation, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden 3School of Medicine and School of Biological Sciences, University of Manchester, Manchester, UK 4Department of Immunology, Georg-August-University Göttingen, Göttingen, Germany 5Research Center for Immunology and Autoimmune Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas-Houston, Houston, TX, USA 6Pulmonary Division, Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Box 440, 405 30 Göteborg, Sweden. Tel.: +46 31 773 3581; Fax: +46 31 416 108; E-mail: [email protected] The EMBO Journal (2006)25:1364-1374https://doi.org/10.1038/sj.emboj.7601004 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Through its involvement in inflammation, opsonization, and cytolysis, the complement protects against infectious agents. Although most of the complement proteins are synthesized in the central nervous system (CNS), the role of the complement system in the normal or ischemic CNS remains unclear. Here we demonstrate for the first time that neural progenitor cells and immature neurons express receptors for complement fragments C3a and C5a (C3a receptor (C3aR) and C5a receptor). Mice that are deficient in complement factor C3 (C3−/−) lack C3a and are unable to generate C5a through proteolytic cleavage of C5 by C5-convertase. Intriguingly, basal neurogenesis is decreased both in C3−/− mice and in mice lacking C3aR or mice treated with a C3aR antagonist. The C3−/− mice had impaired ischemia-induced neurogenesis both in the subventricular zone, the main source of neural progenitor cells in adult brain, and in the ischemic region, despite normal proliferative response and larger infarct volumes. Thus, in the adult mammalian CNS, complement activation products promote both basal and ischemia-induced neurogenesis. Introduction The complement system, a component of the humoral immune system, is involved in inflammation, opsonization, and cytolysis. Many cell types produce or express complement proteins. In the central nervous system (CNS), astrocytes, microglia, and nerve cells are, together, capable of synthesizing or expressing most of the complement proteins (Spiegel et al, 1998; Thomas et al, 2000; D'Ambrosio et al, 2001). However, the role of the complement system in the normal CNS remains unclear. Since the principal manifestation of complement deficiency in the CNS is increased susceptibility to meningococcal meningitis, it has been suggested that these proteins mainly provide defense against infectious agents (Spiegel et al, 1998). C3a and C5a, generated upon complement activation through proteolytic cleavage of the third complement component (C3) and fifth complement component (C5), respectively, are small polypeptides with potent anaphylatoxic properties. C3a and C5a exert their functions at picomolar to nanomolar concentrations by binding to specific receptors, C3a receptor (C3aR) and C5a receptor (C5aR), respectively, that are members of the rhodopsin family of seven-transmembrane G-protein-coupled receptors (Ember et al, 1998). C3a and C5a are both chemoattractant molecules, but whereas C5a has broad proinflammatory effects, the effects of C3a are more selective and rather anti-inflammatory (Ember et al, 1998). Both C3aR and C5aR are expressed in the normal uninjured CNS, predominantly by hippocampal and cortical neurons (Davoust et al, 1999; O'Barr et al, 2001). In vitro studies have shown that C5a is mitogenic for undifferentiated human neuroblastoma cells, whereas it is neuroprotective for terminally differentiated cells (O'Barr et al, 2001). Oligodendrocyte progenitors express C5aR in vitro, but C5aR mRNA expression is downregulated upon differentiation into mature oligodendrocytes (Nataf et al, 2001). The complement system has recently been proposed to participate in tissue regeneration. In amphibians, C3 expression was detected in regenerating limbs but not in developing limbs (Del Rio-Tsonis et al, 1998). The C3 and C5 proteins are expressed in regenerating newt limb and lens (Kimura et al, 2003). The newt ortholog of CD59, a membrane regulator of the terminal complement pathway, has been implicated in blastema positional identity during adult limb regeneration (Morais da Silva et al, 2002). In a mammalian system, C3a and C5a are critical for hepatocyte proliferation and liver regeneration (Mastellos et al, 2001; Strey et al, 2003; Daveau et al, 2004). Finally, C3a promotes homing (Reca et al, 2003), chemotaxis (Honczarenko et al, 2005), and retention of hematopoietic stem and progenitor cells in the bone marrow (Ratajczak et al, 2004), although the issue of C3aR expression on these cells and its involvement in these phenomena remains controversial. We investigated whether neural stem cells express receptors for complement-derived anaphylatoxins and whether complement activation products might play a role in adult mammalian neurogenesis. We show that neural stem cells and neural progenitor cells express C3aR and C5aR, and that the complement system positively regulates basal adult neurogenesis as well as the number of newly born neurons after cerebral ischemia. Results C3aR and C5aR are expressed on neural stem cells and neural progenitors C3aR has been shown to be expressed by hematopoietic stem cells and to play a role in homing to bone marrow (Reca et al, 2003). Oligodendrocyte progenitors express C5aR in vitro (Nataf et al, 2001). To investigate whether complement-derived anaphylatoxin receptors are present on neural stem cells, we stained clonally derived neural stem cells from adult rat hippocampus with antibodies against C3aR and C5aR. Both receptors appeared to be localized in the cell membrane and were homogeneously distributed (Figure 1A and C). As demonstrated by nestin positivity, the cells retained their stem cell phenotype (Figure 1A and C). The specificity of the anti-C3aR and anti-C5aR antibodies, respectively, was confirmed by the absence of immunostaining on brain sections from mice deficient in C3aR (Kildsgaard et al, 2000) and C5aR (Hopken et al, 1996), respectively (Figure 2A and B). Figure 1.Expression of C3aR and C5aR on cultured neural stem cells. Immunostaining of clonally derived neural stem cells from adult rat hippocampus for C3aR (A) and C5aR (C) shows homogeneous distribution of both receptors in the cell membrane. Nestin immunostaining confirmed that the cells retain their stem cell phenotype. The cells were infected with retrovirus to express green fluorescent protein (GFP). Negative control immunostaining, in which the primary antibodies against C3aR, C5aR, and nestin were omitted (B, D). Scale bar equals 20 μm. Download figure Download PowerPoint Figure 2.Expression of C3aR and C5aR on transit-amplifying precursors and migrating neuroblasts in vivo. Immunostaining of paraffin-embedded brain sections from adult mice with antibodies against C3aR (A) and C5aR (B). Both receptors are expressed by cells in the SVZ and rostral migratory stream of WT mice. The specificity of the anti-C3aR and anti-C5aR antibodies, respectively, was confirmed by the absence of immunostaining on brain sections from C3aR−/− and C5aR−/−mice. In the negative control immunostainings of WT brain sections, the primary antibodies were omitted. Olig2-positive transit-amplifying precursor cells in the SVZ are colabeled by anibodies against C3aR (C) and C5aR (D). Dcxpos migrating neuroblasts in the rostral migratory stream express C3aR (E) and C5aR (F). Arrows indicate double-labeled cells in the insert. Scale bars equal 50 μm (A, B) 10 μm (C, D) and 60 μm (E, F). Download figure Download PowerPoint Oligodendrocyte transcription factor 2 (Olig2) is a marker of fast-dividing transit-amplifying precursor cells in the subventricular zone (SVZ) (Hack et al, 2004, 2005). Using brain sections from wild-type (WT) C57BL/6 mice, we showed that these cells express C3aR and C5aR (Figure 2C and D). Colabeling with doublecortin (Dcx), a marker of migrating neuroblasts, which are normally present in the SVZ and rostral migratory stream of adult brain (Nacher et al, 2001), showed that, in both regions, all Dcx-positive (Dcxpos) cells were also positive for C5aR and many were positive for C3aR (Figure 2E and F). Immunostaining for NeuN, a marker of differentiated neurons, showed that over 95% of NeuNpos cells were also positive for the C3aR and C5aR, whereas we did not find any colocalization of C3aR or C5aR with glial fibrillary acidic protein (GFAP), which marks adult neural stem cells as well as mature astrocytes (Doetsch et al, 1999) (data not shown). Thus, neural stem cells in vitro as well as murine neural progenitor cells, migrating neuroblasts, and mature neurons in vivo express C3aR and C5aR. Reduced basal neurogenesis in the absence of signaling through C3aR To assess whether signaling through the C3aR might play a role in basal neurogenesis, we injected WT C57BL/6 mice with a nonpeptide antagonist of the C3aR, SB290157 (Ames et al, 2001) for 10 days. Control as well as C3−/− mice were injected with vehicle. For the first 7 days, the mice also received bromodeoxyuridine (BrdU). Proliferating neural stem cells (GFAPposBrdUpos), transit-amplifying cells (Olig2posBrdUpos), migrating neuroblasts (DcxposBrdUpos), newly formed neurons (NeuNposBrdUpos), and BrdUpos cells were counted in the two principal sites of adult neurogenesis, SVZ and the dentate gyrus subgranular zone (SGZ) of the hippocampus, in the dentate gyrus granule cell layer (GCL) as well as in the olfactory bulb (OB), the final destination for the neuroblasts originating in SVZ under basal conditions (Gage, 2000; Alvarez-Buylla and Garcia-Verdugo, 2002; Doetsch, 2003). The number of DcxposBrdUpos cells in OB and SGZ was reduced by 33–34% in both the C3−/− mice (P<0.0005) and the mice that received the C3aR antagonist compared to control mice (P<0.0005) (Figure 3). The quantification of NeuNposBrdUpos cells in OB and SGZ/GCL showed a 22–25% reduction in the number of these cells in both the C3−/− mice (P<0.05 and <0.005, respectively) and the C3aR antagonist-treated mice (P<0.005) (Figure 4). The number of BrdUpos, GFAPposBrdUpos, and Olig2posBrdUpos cells in any of the regions was not affected by the C3 deficiency or C3aR antagonist treatment (data not shown). Figure 3.Proliferating migrating neuroblast in the dentate gyrus SGZ visualized by confocal microscopy in an orthogonal projection composed of 14 optical z-planes, 0.5 μm thick (A). The number of DcxposBrdUpos cells in SGZ, SVZ, and OB in control mice (n=12), C3−/− mice (n=6), and mice treated with C3aR antagonist (n=10) (B). Representative low-power images of DcxposBrdUpos cells in SGZ, SVZ, and OB in the three groups of mice; Dcx, green, BrdU, red (C). Values are the number of cells per region. *P<0.05, **P<0.0005. Scale bars equal 10 μm (A) and 200 μm (C). Download figure Download PowerPoint Figure 4.A newly formed neuron in the dentate gyrus GCL visualized by confocal microscopy in an orthogonal projection composed of 14 optical z-planes, 0.5 μm thick (A). The number of NeuNposBrdUpos cells in the SGZ/GCL and OB in control mice (n=12), C3−/− mice (n=6) and mice treated with C3aR antagonist (n=10) (B). Values are the number of cells per region. *P<0.05, **P<0.005. Scale bar equals 10 μm. Download figure Download PowerPoint To further confirm the role of C3aR in neurogenesis, we injected C3aR−/− and WT C57BL/6 mice with BrdU for 7 days and assessed the number of BrdUpos, DcxposBrdUpos, and NeuNposBrdUpos cells in SVZ, OB, and SGZ/GCL 14 days later. In the C3aR−/− mice, the number of DcxposBrdUpos cells in OB and SGZ was 22 and 28% lower, respectively (P<0.05) (Figure 5A). Similarly, the C3aR−/− mice showed a 22–28% reduction in the number of NeuNposBrdUposcells in OB and SGZ/GCL (P<0.05), (Figure 5B). There was no difference in the number of BrdUpos cells between the groups (data not shown). Figure 5.Basal neurogenesis is reduced in C3aR−/− mice. Compared to control (WT) mice (n=6), the C3aR−/− mice (n=6) have lower number of newly formed migrating neuroblasts in the SGZ and OB (A) and lower number of newly formed neurons in SGZ/the dentate gyrus GCL and OB (B). Values are the number of cells per region. *P<0.05. The higher number of NeuNposBrdUpos cells in control mice in this figure compared to Figure 4 is likely due to a difference in survival time after the last BrdU injection (14 versus 3 days). Download figure Download PowerPoint Taken together, these findings indicate that signaling through the C3aR positively regulates basal neurogenesis, presumably by stimulating differentiation of neural progenitors. Since the newly formed cells have to migrate over a considerable distance from SVZ to their final destination in OB, the observed findings in OB can be the result of a combined defect in cell migration and differentiation in the absence of C3aR signaling. Reduced ischemia-induced neurogenesis in SVZ in C3−/− mice C3−/− mice lack C3a and are unable to generate C5a through proteolytic cleavage of C5 by C5-convertase (Pekna et al, 1998). To determine if complement activation plays a role in ischemia-triggered neurogenesis, we subjected C3−/− mice and WT controls to middle cerebral artery (MCA) occlusion (MCAO), a model of cerebral ischemia that leads to an ischemic infarct in the cerebral cortex and striatum. After ischemia, neural progenitor cells were reported to proliferate in the SVZ ipsilateral to the insult, migrate into the damaged area, and differentiate into neurons (Arvidsson et al, 2002; Parent et al, 2002; Jin et al, 2003). Therefore, we compared the numbers of Dcxpos migrating neuroblasts in the SVZ in the two groups of mice 7 days after ischemia. We found that the WT mice had 46% more Dcxpos cells in the ipsilateral than in the contralateral SVZ (P<0.005) compared to increase by 26% in the C3−/− mice (P=0.064). Moreover, the C3−/− mice had 24% fewer Dcxpos cells in the ipsilateral SVZ than WT mice (P<0.05); both groups had a similar number of these cells in the contralateral SVZ (Figure 6A). Figure 6.Number of Dcxpos migrating neuroblasts in the SVZ in the ipsilateral (I) and contralateral (C) hemispheres of WT (n=7, 8, 8) and C3−/− (n=9, 7, 11) mice. The mice were subjected to focal cerebral ischemia by MCAO or MCAT. Dcxpos cells in the SVZ were counted in three sections per mouse 7 days after MCAO (A) and MCAT (B) and 21 days after MCAT (C). Values are the number of cells per section. *P<0.05, **P<0.01, ***P<0.005. Download figure Download PowerPoint To confirm the findings from the MCAO study and to be able to assess ischemia-induced neurogenesis at a later time point, we used a MCA transection (MCAT) model in which the infarct volume is approximately 20% of that after MCAO (Welsh et al, 1987; Fotheringham et al, 2000). At both 7 and 21 days after MCAT, the WT mice had 38% more Dcxpos cells in the ipsilateral than in the contralateral SVZ (P<0.01 and <0.05, respectively). Interestingly, the C3−/− mice showed a less pronounced ischemia-induced increase in the number of Dcxpos cells with only 22 and 27%, respectively, more Dcxpos cells in the ipsilateral compared to the contralateral SVZ (P=0.2 and <0.05, respectively), (Figure 6B and C). These data suggest that the complement acts as a positive regulator of the ischemia-induced neural progenitor differentiation in SVZ. Alternatively, complement activation products may stimulate neural progenitor proliferation after ischemia. To distinguish between these two possibilities, we evaluated cell proliferation in SVZ using in vivo BrdU labeling. Before and for 7 days after MCAT, mice were injected with BrdU. In both groups, BrdUpos cells were more numerous in the ipsilateral than in the contralateral SVZ at 7 days (92.8±4.0 versus 77.9±4.1, P<0.05 for C3−/− mice and 90.0±3.9 versus 70.7±4.4, P<0.005 for WT mice) and 21 days after MCAT (14.5±1.0 versus 10.6±0.8, P<0.01 and 16.3±1.5 versus 10.9±1.1, P<0.05). Thus, complement stimulates neural progenitor differentiation in SVZ after ischemia. Reduced ischemia-induced neurogenesis in the penumbra and infarct area in C3−/− mice To determine whether complement affects the number of neural progenitor cells in the penumbra and infarct area, we stained the sections for nestin, a marker of neural progenitor cells that is also expressed by reactive astrocytes (Lendahl et al, 1990), and glial fibrillary protein (GFAP), a marker of reactive astrocytes. Compared with controls, the C3−/− mice had 50% fewer nestinpos GFAPneg neural progenitor cells in the infarct area (3.6±0.97 versus 8.3±1.76 cells/section, P<0.05) and in the penumbra (15.9±2.02 versus 28.4±2.22 cells/section, P<0.005) 7 days after MCAO and 30% fewer nestinpos GFAPneg cells in the penumbra (11.3±0.2 versus 16.1±0.5 cells/10 mm2, P<0.00001) 7 days after MCAT (Figure 7). Furthermore, the fraction of GFAPneg cells among the nestinpos cells was reduced by 29% in the C3−/− mice (5.4±0.1 versus 7.6±0.2%, P<0.00001). No nestinpos cells were detected in the penumbra of either group of mice at 21 days. It is not clear whether these cells are locally derived or originate in the neurogenic SVZ, as there is evidence to support both alternatives (Reynolds and Weiss, 1992; Palmer et al, 1999; Kondo and Raff, 2000; Gould et al, 2001). Regardless of their origin, the response of neural progenitors in the penumbra is positively regulated by complement-derived stimuli. Figure 7.Neural progenitor cells in the infarct area and penumbra of WT (n=7, 8, 8) and C3−/− (n=9, 7, 11) mice after focal cerebral ischemia. Immunostaining for nestin and GFAP was used to visualize nestinpos GFAPneg neural progenitor cells (A). These cells were counted in 2–3 sections/mouse in the infarct area and penumbra 7 days after MCAO and in the penumbra 7 days after MCAT (B). Values are the number of cells per section (MCAO) and the number of cells/10 mm2 (MCAT). Representative low-power images of the penumbra in the two groups of mice; nestin, red, GFAP, green. Arrows indicate nestinpos GFAPneg cells. Broken line depicts the infarct border. i, infarct (C). *P<0.05, ***P<0.005, *****P<0.00001. Scale bars equal 15 μm (A) and 200 μm (C). Download figure Download PowerPoint To address whether the reduction in the number of neural progenitor cells and migrating neuroblasts might lead to reduced formation of new neurons, we used combined immunostaining for NeuN and BrdU. Intriguingly, the C3−/− mice had fewer newly formed neurons (NeuNposBrdUpos cells) in the penumbra both 7 days (7.5±0.4 versus 10.0±0.5 cells/10 mm2, P<0.001) and 21 days after MCAT (9.7±0.6 versus 13.3±0.9 cells/10 mm2, P<0.005) (Figure 8). Also, the fraction of NeuNpos cells among the BrdUpos cells was reduced in the C3−/− mice (3.4±0.2 versus 4.76±0.3%, P<0.005 and 4.9±0.4 versus 6.5±0.4%, P<0.05). Figure 8.Ischemia-induced generation of new neurons. A newly formed NeuNposBrdUpos neuron in the penumbra visualized by confocal microscopy in an orthogonal projection composed of 14 optical z-planes, 0.5 μm thick (A). Number of NeuNposBrdUpos cells in the penumbra of WT (n=10, 11) and C3−/− (n=9, 11) mice 7 and 21 days after MCAT (B). The number of NeuNposBrdUpos cells/10 mm2 was counted in the penumbra on 2–3 sections/mouse. ***P<0.005, ****P<0.001. Scale bar equals 15 μm. Download figure Download PowerPoint Postmitotic neurons can incorporate BrdU through stress-induced cell cycle activation before undergoing delayed neuronal death by apoptosis (Sanes and Okun, 1972; Osuga et al, 2000; Katchanov et al, 2001; Liu and Greene, 2001; Verdaguer et al, 2002). To determine whether the NeuNposBrdUpos cells in the penumbra are undergoing ischemia-induced apoptosis, we performed triple-label immunostaining with antibodies against BrdU, NeuN, and activated caspase-3, the latter being expressed in association with delayed ischemic neuronal death (Namura et al, 1998). No activated caspase-3pos cells were detected in the infarct area or within the penumbra 21 days after MCAT; however, 7 days after MCAT, caspase-3 immunoreactivity was confined to the infarct and NeuN to the penumbra (Figure 9). No NeuNposBrdUpos cells were also positive for activated caspase-3, which proves that the NeuNposBrdUpos cells were not undergoing caspase-3-dependent apoptosis and supports the argument that they are neurons newly formed after the ischemic injury. Thus, even in the ischemic region, complement activation-derived signals positively regulate the number of both neural progenitor cells and newly formed neurons. Figure 9.The NeuNposBrdUpos cells are not undergoing ischemia-induced apoptosis. Immunostaining for BrdU, NeuN and caspase-3 on day 7 (A) and day 21 (B) after MCAT. Broken line depicts the infarct border. p, penumbra; i, infarct. Higher magnification images of boxed areas in (A) showing penumbra (P) and infarct (I). Scale bar equals 120 μm (A, B) and 60 μm (P, I). Download figure Download PowerPoint No activated caspase-3pos cells were detected in the SVZ in either of the groups (not shown). It is therefore unlikely that increased cell death accounts for the observed difference in the number of Dcxpos cells or that the complement-derived signals affect the survival of neural progenitor cells. Impaired ischemia-induced neurogenesis in C3−/− mice is not the consequence of reduced infarct size Earlier studies using a transient cerebral ischemia model have shown that inhibition of complement activation reduced infarction volume (Akita et al, 2003; De Simoni et al, 2003). To determine whether the reduced ischemia-induced neurogenic response in the C3−/− mice is the consequence of reduced production or release of neurogenic factors due to reduced ischemic damage, we evaluated the brain tissue volume lost to infarction. We found that the cerebral infarct volume was 24% larger in C3−/− mice than in the WT mice (16.3±0.31 versus 13.1±1.07 mm3, P<0.01) 7 days after MCAO. At 7 days after MCAT the infarct volume did not significantly differ between the groups, although there was a tendency toward larger volume in the C3−/− mice (2.5±0.5 versus 1.6±0.3 mm3, P=0.14). However, 21 days after MCAT, the C3−/− mice had lost twice as much tissue as controls (2.3±0.2 versus 1.1±0.2 mm3, P<0.0005). The neuronal density, as assessed by the quantification of NeuNpos cells, in the penumbra 7 days after ischemia did not differ between the C3−/− and control mice (1393±78.1 and 1330±92.3 cells/10 mm2) (Figure 10). Figure 10.Representative low-power images of the density of neurons (NeuNpos), reactive astrocytes (GFAPpos), and microglia (isolectinpos) at the infarct border in C3−/− (A) and WT (B) mice 7 days after cerebral ischemia. The sections were stained with antibodies against NeuN and GFAP, and with isolectin. Broken line depicts the infarct border. i, infarct. Scale bars equal 150 μm. Download figure Download PowerPoint We also assessed the activation of astrocytes and recruitment of microglia. Immunostaining for GFAP 7 days after ischemia showed a massive presence of highly GFAPpos astrocytes at the infarct border in both C3−/− and WT mice. Similarly, isolectin staining that identifies microglia and endothelial cells demonstrated a rim of activated microglial cells around the ischemic lesion and revealed migration of these cells into the infarct area in both groups of mice. The width of the GFAPpos band was comparable in C3−/− and WT mice (456±62.3 and 440±25.1 μm), as was the density of the GFAPpos cells (824±28.0 and 729±77.0 cells/10 mm2) (Figure 10). Similarly, there was no difference in width of the isolectinpos band (367±18.8 and 319±20.3 μm) or the density of isolectinpos cells (142±28.0 and 173±26.4 cells/10 mm2) between the groups (Figure 10). The data imply that impaired neurogenesis observed in the C3−/− mice cannot be explained by reduced availability of complement-independent neurogenic stimuli due to smaller ischemic damage, and that the lack of C3 is not associated with impaired activation of astrocytes or microglia. Discussion It has been demonstrated that essentially all of the activation components, regulatory molecule
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