Population Control of Resident and Immigrant Microglia by Mitosis and Apoptosis
2007; Elsevier BV; Volume: 171; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.061044
ISSN1525-2191
AutoresMartin Wirenfeldt, Lasse Dissing‐Olesen, Alicia A. Babcock, Marianne Nielsen, Michael Meldgaard, Jens Zimmer, Íñigo Azcoitia, Robert Graham Quinton Leslie, Frederik Dagnæs‐Hansen, Bente Finsen,
Tópico(s)Barrier Structure and Function Studies
ResumoMicroglial population expansion occurs in response to neural damage via processes that involve mitosis and immigration of bone marrow-derived cells. However, little is known of the mechanisms that regulate clearance of reactive microglia, when microgliosis diminishes days to weeks later. We have investigated the mechanisms of microglial population control in a well-defined model of reactive microgliosis in the mouse dentate gyrus after perforant pathway axonal lesion. Unbiased stereological methods and flow cytometry demonstrate significant lesion-induced increases in microglial numbers. Reactive microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations. Microglial population expansion occurs in response to neural damage via processes that involve mitosis and immigration of bone marrow-derived cells. However, little is known of the mechanisms that regulate clearance of reactive microglia, when microgliosis diminishes days to weeks later. We have investigated the mechanisms of microglial population control in a well-defined model of reactive microgliosis in the mouse dentate gyrus after perforant pathway axonal lesion. Unbiased stereological methods and flow cytometry demonstrate significant lesion-induced increases in microglial numbers. Reactive microglia often occurred in clusters, some having recently incorporated bromodeoxyuridine, showing that proliferation had occurred. Annexin V labeling and staining for activated caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that apoptotic mechanisms participate in dissolution of the microglial response. Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable. Microglial numbers and responses were severely reduced in bone marrow chimeric mice. These results broaden our understanding of the microglial response to neural damage by demonstrating that simultaneously occurring mitosis and apoptosis regulate expansion and reduction of both resident and immigrant microglial cell populations. Microglia represent the first line of defense against pathogens or injury in the central nervous system (CNS).1Kreutzberg GW Microglia: a sensor for pathological events in the CNS.Trends Neurosci. 1996; 19: 312-318Abstract Full Text Full Text PDF PubMed Scopus (3811) Google Scholar By dynamically surveying the CNS, microglia serve important maintenance functions for neurons, with which they have intimate contact.2Nimmerjahn A Kirchhoff F Helmchen F Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.Science. 2005; 308: 1314-1318Crossref PubMed Scopus (4197) Google Scholar, 3Davalos D Grutzendler J Yang G Kim JV Zuo Y Jung S Littman DR Dustin ML Gan WB ATP mediates rapid microglial response to local brain injury in vivo.Nat Neurosci. 2005; 8: 752-758Crossref PubMed Scopus (2891) Google Scholar Key functions include metabolism of nucleosides and purines,2Nimmerjahn A Kirchhoff F Helmchen F Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.Science. 2005; 308: 1314-1318Crossref PubMed Scopus (4197) Google Scholar, 3Davalos D Grutzendler J Yang G Kim JV Zuo Y Jung S Littman DR Dustin ML Gan WB ATP mediates rapid microglial response to local brain injury in vivo.Nat Neurosci. 2005; 8: 752-758Crossref PubMed Scopus (2891) Google Scholar phagocytosis, and production of growth factors and cytokines.4Streit WJ Walter SA Pennell NA Reactive microgliosis.Prog Neurobiol. 1999; 57: 563-581Crossref PubMed Scopus (1027) Google Scholar, 5Aloisi F Immune function of microglia.Glia. 2001; 36: 165-179Crossref PubMed Scopus (1077) Google Scholar The significance of microglial function in maintaining CNS homeostasis is evident from the discovery that individuals with loss of function mutations of the TREM2/DAP12 receptor complex, which renders microglia unable to phagocytose apoptotic neurons in mice,6Takahashi K Rochford CDP Neumann H Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2.J Exp Med. 2005; 201: 647-657Crossref PubMed Scopus (816) Google Scholar develop symptoms of presenile dementia.7Paloneva J Kestila M Wu J Salminen A Bohling T Ruotsalainen V Hakola P Bakker ABH Phillips JH Pekkarinen P Lanier LL Timonen T Peltonen L Loss of function mutations in TYROBP (DAP12) result in a presenile dementia with bone cysts.Nat Genet. 2000; 25: 357-361Crossref PubMed Scopus (377) Google Scholar Microglia are unique cells in the CNS parenchyma, being innate immune cells and having a mesodermal origin. In line with their myeloid lineage,8del Rio-Hortega P Microglia.in: Penfield W Cytology and Cellular Pathology of the Nervous System. Paul B. Hoeber, New York1932: 481-534Google Scholar slow microglial turnover in normal CNS9Lawson LJ Perry VH Gordon S Turnover of resident microglia in the normal adult-mouse brain.Neuroscience. 1992; 48: 405-415Crossref PubMed Scopus (565) Google Scholar and enhanced recruitment during reactive microgliosis10Priller J Flugel A Wehner T Boentert M Haas CA Prinz M Fernandez-Klett F Prass K Bechmann I de Boer BA Frotscher M Kreutzberg GW Persons DA Dirnagl U Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment.Nat Med. 2001; 7: 1356-1361Crossref PubMed Scopus (538) Google Scholar, 11Wirenfeldt M Babcock AA Ladeby R Lambertsen KL Dagnaes-Hansen F Leslie RG Owens T Finsen B Reactive microgliosis engages distinct responses by microglial subpopulations after minor central nervous system injury.J Neurosci Res. 2005; 82: 507-514Crossref PubMed Scopus (54) Google Scholar indicate that microglia are potentially replaceable cells, making microglia and their myeloid progenitors promising candidates for active cellular therapy in CNS disease and injury.12Biffi A De Palma M Quattrini A Del Carro U Amadio S Visigalli I Sessa M Fasano S Brambilla R Marchesini S Bordignon C Naldini L Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells.J Clin Invest. 2004; 113: 1118-1129Crossref PubMed Scopus (264) Google Scholar, 13Asheuer M Pflumio FO Benhamida S Dubart-Kupperschmitt A Fouquet F Imai Y Aubourg P Cartier N Human CD34(+) cells differentiate into microglia and express recombinant therapeutic protein.Proc Natl Acad Sci USA. 2004; 101: 3557-3562Crossref PubMed Scopus (137) Google Scholar For potential microglial cell therapy to be safe and applicable for the treatment of neurological disease, information is needed about the population control of immigrating microglia that become involved in the microglial reaction. Acute activation of microglia as a result of neural injury or pathology quickly leads to reactive microgliosis, a cardinal feature being expansion in the number of microglia in the affected region. Increase in cell number originates in part from recruitment of myeloid cells,11Wirenfeldt M Babcock AA Ladeby R Lambertsen KL Dagnaes-Hansen F Leslie RG Owens T Finsen B Reactive microgliosis engages distinct responses by microglial subpopulations after minor central nervous system injury.J Neurosci Res. 2005; 82: 507-514Crossref PubMed Scopus (54) Google Scholar proliferation,14Hailer NP Grampp A Nitsch R Proliferation of microglia and astrocytes in the dentate gyrus following entorhinal cortex lesion: a quantitative bromodeoxyuridine-labelling study.Eur J Neurosci. 1999; 11: 3359-3364Crossref PubMed Scopus (96) Google Scholar or migration from juxtaposed regions.15Rappert A Bechmann I Pivneva T Mahlo J Biber K Nolte C Kovac AD Gerard C Boddeke HWGM Nitsch R Kettenmann H CXCR3-dependent microglial recruitment is essential for dendrite loss after brain lesion.J Neurosci. 2004; 24: 8500-8509Crossref PubMed Scopus (218) Google Scholar The state of reactive microgliosis dissolves days to weeks later, according to an inherently tightly regulated schedule, which has been suggested to involve microglial apoptosis.16Jones LL Banati RB Graeber MB Bonfanti L Raivich G Kreutzberg GW Population control of microglia: does apoptosis play a role?.J Neurocytol. 1997; 26: 755-770Crossref PubMed Scopus (60) Google Scholar The cellular population control of immigrating and resident microglia should be comparable if immigrating bone marrow (BM)-derived cells are to take part fully in regular microglial tasks. To address these fundamental questions, we investigated whether resident and immigrating microglia are governed by similar mechanisms of population control, ie, cellular multiplication by mitosis and reduction of the cellular population by apoptosis. Reactive microgliosis was induced in the dentate gyrus and hippocampus in unmanipulated and green fluorescent protein (GFP)-BM-chimeric mice by transection of the perforant pathway (PP) projection in the entorhinal cortex. Our results show that microglial expansion is balanced by simultaneously occurring mitosis and apoptosis. The axonal lesion-induced mitotic activity of resident microglia supersedes that of BM-derived immigrant microglia, whereas the kinetics of lesion-induced apoptotic responses are comparable. C57BL/6J mice (Taconic, Skensved, Denmark; or Harlan, Allerød, Denmark) were used for studies of nonchimeric mice. For BM-chimeric studies, C57BL/6 congenic B6.SJL-PtprcaPepcb/BoyJ mice (The Jackson Laboratory, Bar Harbor, ME), which express the CD45.1 allotype and are hence abbreviated B6(CD45.1), were used as transplant recipients and C57BL/6-Tg(UBC-GFP)30Scha/J mice (The Jackson Laboratory)17Schaefer BC Schaefer ML Kappler JW Marrack P Kedl RM Observation of antigen-dependent CD8+ T-cell/dendritic cell interactions in vivo.Cell Immunol. 2001; 214: 110-122Crossref PubMed Scopus (324) Google Scholar as BM donors. This combination of mice was chosen so that immigrating cells could be identified in chimeric mice by two criteria: positive expression of GPF and CD45.2, which is normally expressed in C57BL/6 mice. Expression of the GFP transgene, however, proved by itself to be a sufficiently strong and reliable signal. A group of nonirradiated B6(CD45.1) mice (The Jackson Laboratory) was included to control for potential differences from C57BL/6 mice. Mice were housed and bred and animal experiments performed at the Laboratory of Biomedicine at the University of Southern Denmark and at the Department of Medical Microbiology and Immunology at Aarhus University. All animal experiments were performed according to Danish law and protocols approved by the Danish Ethical Animal Care Committee. Microglial activation in the hippocampus and dentate gyrus was induced by the anterograde axonal and terminal degeneration of presynaptic elements resulting from transection of the entorhino-hippocampal PP projection,18Hjorth-Simonsen A Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata.J Comp Neurol. 1972; 146: 219-232Crossref PubMed Scopus (418) Google Scholar, 19Fifková E Two types of terminal degeneration in molecular layer of dentate fascia following lesions of entorhinal cortex.Brain Res. 1975; 96: 169-175Crossref PubMed Scopus (50) Google Scholar, 20Matthews DA Cotman C Lynch G Electron-microscopic study of lesion-induced synaptogenesis in dentate gyrus of adult rat. 1. Magnitude and time course of degeneration.Brain Res. 1976; 115: 1-21Crossref PubMed Scopus (390) Google Scholar, 21Amaral DG Witter MP Hippocampal formation.in: The Rat Nervous System. Academic Press, Inc., San Diego1995: 443-493Google Scholar performed as previously described.11Wirenfeldt M Babcock AA Ladeby R Lambertsen KL Dagnaes-Hansen F Leslie RG Owens T Finsen B Reactive microgliosis engages distinct responses by microglial subpopulations after minor central nervous system injury.J Neurosci Res. 2005; 82: 507-514Crossref PubMed Scopus (54) Google Scholar Mice were anesthetized with a mixture of ketamine and xylazine (for flow cytometry) or fentanyl citrate, fluanisone, and diazepam (for histology) and fixed in a stereotaxic frame (Stoelting, Wood Dale, IL). The PP transection was made with a wire knife (David Kopf Instruments, Tujunga, CA) angled 10 degrees lateral and rotated 15 degrees rostral and the nosebar set at −3 mm. The closed wire knife was inserted 2.1 mm lateral to the lambda and 0.3 mm caudal to the lambdoid suture through a drilled trepanation. A 3.1-mm-long cut was made in the entorhinal cortex starting 3.4 mm ventral to the meninges. Mice were supplied with eye ointment and postoperative injections of saline and buprenorphine. Animals were placed in a warm environment for postoperative recovery. Recipient mice were irradiated with 9.5 Gy in a single dose from a 137Cs source (Risø National Laboratory, Roskilde, Denmark) and were reconstituted with BM cells obtained from GFP transgenic donor mice.10Priller J Flugel A Wehner T Boentert M Haas CA Prinz M Fernandez-Klett F Prass K Bechmann I de Boer BA Frotscher M Kreutzberg GW Persons DA Dirnagl U Targeting gene-modified hematopoietic cells to the central nervous system: use of green fluorescent protein uncovers microglial engraftment.Nat Med. 2001; 7: 1356-1361Crossref PubMed Scopus (538) Google Scholar, 11Wirenfeldt M Babcock AA Ladeby R Lambertsen KL Dagnaes-Hansen F Leslie RG Owens T Finsen B Reactive microgliosis engages distinct responses by microglial subpopulations after minor central nervous system injury.J Neurosci Res. 2005; 82: 507-514Crossref PubMed Scopus (54) Google Scholar Donor BM cells were harvested by flushing the medullary canal of the femoral and tibial diaphysis with RPMI 1640 medium (Gibco, Paisley, UK). After centrifugation, the cell suspension was filtered and transplanted by intravenous injection. The transplanted mice were supplied with oxytetracycline (2 g/L Terramycin veterinary 20%; Pfizer, Amoise, France) in the drinking water for 3 days after transplantation.11Wirenfeldt M Babcock AA Ladeby R Lambertsen KL Dagnaes-Hansen F Leslie RG Owens T Finsen B Reactive microgliosis engages distinct responses by microglial subpopulations after minor central nervous system injury.J Neurosci Res. 2005; 82: 507-514Crossref PubMed Scopus (54) Google Scholar Chimerism was assessed by expression of GFP in blood cells at the time of sacrifice. Positive GFP expression was determined using autofluorescence levels in blood cells from non-Tg mice as negative controls. Flow cytometric analysis showed that 91.6 ± 1.1% (mean ± SEM) of blood cells were GFP+, indicating that successful reconstitution had occurred. Genomic DNA was extracted from external ear tissue, obtained from labeling of animals for identity, by alkaline lysis in 0.1 mol/L KOH for 1 hour at 95°C followed by KH2PO4 neutralization. Extracts were diluted 20-fold, and 2 μl were applied for 25-μl real-time PCR. The reaction mixture contained 1× RealQ master (Ampliqon; Bie and Berntsen A/S, Rødovre, Denmark) and for transgene PCRs 300 nmol/L of each of the two primers (5′-ATTGTCCGCTAAATTCTGGCCGTTT/GCTCGACCAGGATGGGCACC-3′) (TAG, Copenhagen, Denmark) and SYBR Green diluted 20,000-fold. Lymphotoxin-α (LT-α) reference gene PCRs contained 400 nmol/L of the two primers (5′-GTCCAGCTCTTTTCCTCCCAAT/GTCCTTGAAGTCCCGGATACAC-3′) and 50 nmol/L TaqMan probe (5′-CCTTCCATGTGCCTCTCCTCAGTGCG-3′). For PCR and detection in real time, an iCycler (Bio-Rad, Herlev, Denmark) was used. The thermocycling protocol was 95°C for 15 minutes to activate the RealQ enzyme followed by 40 cycles of 95°C for 15 seconds, 62°C for 30 seconds, and 72°C for 45 seconds. RNA was extracted using RNeasy Protect mini kits (Qiagen, Hilden, Germany) or TRIzol (Invitrogen, Taastrup, Denmark), as previously described for PP-lesioned hippocampus samples.22Babcock AA Kuziel WA Rivest S Owens T Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS.J Neurosci. 2003; 23: 7922-7930Crossref PubMed Google Scholar, 23Meldgaard M Fenger C Lambertsen KL Pedersen MD Ladeby R Finsen B Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.J Neurosci Methods. 2006; 156: 101-110Crossref PubMed Scopus (44) Google Scholar Complementary DNA was synthesized from 0.4 μg of total RNA by reverse transcription, using previously established conditions.23Meldgaard M Fenger C Lambertsen KL Pedersen MD Ladeby R Finsen B Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.J Neurosci Methods. 2006; 156: 101-110Crossref PubMed Scopus (44) Google Scholar For real-time PCR, a Bio-Rad iCycler was used. Each reaction contained a total volume of 25 μl, 5 of which were diluted cDNA. The other components were RealQ master mix (1.5 mmol/L magnesium at 1×, Ampliqon; Bie and Berntsen A/S), 1 to 2 mmol/L of additional MgCl2, SYBR Green, fluorescein, and 300 nmol/L of each PCR primer. Newly designed primer sets were validated to specifically amplify the respective target only. These were M-CSF (5′-CGCTGCCCTTCTTCGACATG/ACACCTCCTTGGCAATACTCCT-3′; annealing temperature, 60°C; magnesium concentration, 3.5 mmol/L), M-CSFR (5′-GCATTACAACTGGACCTACCTA/AGAGCTTGAATGTGTACCTGTAT-3′; annealing temperature, 55°C; magnesium concentration, 3.0 mmol/L), GM-CSF, which was targeted by two different primer sets (5′-CATGTAGAGGCCATCAAAGAAG/ACGACTTCTACCTCTTCATTCAA-3′; annealing temperature, 56°C; magnesium concentration, 3.5 mmol/L; and 5′-GGGCAATTTCACCAAACTCAA/TTTCACAGTCCGTTTCCGG-3′; annealing temperature, 56°C; magnesium concentration, 3.5 mmol/L) targeting exons 1/2 and 3/4, respectively, and GM-CSFR (5′-AGTGACGTGCAGGAGGTTCG/ACGTCGTCGGACACCTTGT-3′; annealing temperature, 60°C; magnesium concentration, 3.5 mmol/L). Amplification of CD11b and HPRT1 was done using previously published primer and probe sequences, and FAM- or HEX-labeled TaqMan probes instead of SYBR Green, respectively.23Meldgaard M Fenger C Lambertsen KL Pedersen MD Ladeby R Finsen B Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.J Neurosci Methods. 2006; 156: 101-110Crossref PubMed Scopus (44) Google Scholar Each cDNA was subjected to triplicate real-time PCR analysis. Calibrator samples were included on all plates. Data were nor- malized using HPRT1 reference gene, averaged, and calibrated against the calibrator ratio. Data are presented as relative values. For in vivo detection of microglial mitosis, proliferating cells were labeled with 5-bromo-2′-deoxyuridine (BrdU), which is incorporated into DNA during mitotic S phase. Each mouse was injected with a 90-mg/kg dose of BrdU dissolved in phosphate-buffered saline (PBS; 10 mg/ml) intraperitoneally three times at 8-hour intervals for the last 24 hours before perfusion. Mice used for BrdU histology were injected with 50 mg/kg 1 hour before sacrifice. Mice were sacrificed under pentobarbital anesthesia by exsanguination and intracardiac perfusion with 20 ml of PBS. The brains were removed, and the hippocampus and dentate gyrus were dissected out en bloc from the contralateral and lesioned hemispheres. These samples did not include tissue surrounding the wire knife lesion because this was in the entorhinal cortex, and any adherent choroid plexus was removed.24Babcock AA Wirenfeldt M Holm T Nielsen HH Dissing-Olesen L Toft-Hansen H Millward JM Landmann R Rivest S Finsen B Owens T Toll-like receptor 2 signaling in response to brain injury: an innate bridge to neuroinflammation.J Neurosci. 2006; 26: 12826-12837Crossref PubMed Scopus (160) Google Scholar Hippocampal tissue was homogenized through a 70-μm cell strainer (BD Falcon, Franklin Lakes, NJ) in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco). After centrifugation, cells were incubated with anti-FcγIII/II receptor antibody (BD Biosciences, Erembodegem, Belgium) and 50 μg/ml of Syrian hamster Ig (Jackson Immunoresearch, West Grove, PA) in RPMI 1640 medium with 10% FBS at room temperature for 45 to 60 minutes to block nonspecific staining.11Wirenfeldt M Babcock AA Ladeby R Lambertsen KL Dagnaes-Hansen F Leslie RG Owens T Finsen B Reactive microgliosis engages distinct responses by microglial subpopulations after minor central nervous system injury.J Neurosci Res. 2005; 82: 507-514Crossref PubMed Scopus (54) Google Scholar, 22Babcock AA Kuziel WA Rivest S Owens T Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS.J Neurosci. 2003; 23: 7922-7930Crossref PubMed Google Scholar For annexin V analysis, surface antigen labeling was performed by incubating cells for 30 minutes at room temperature with phycoerythrin (PE)-conjugated anti-CD45 antibody and fluorescein isothiocyanate-conjugated anti-CD11b (BD Biosciences) in RPMI 1640 medium with 10% FBS. For studies in chimeric mice, anti-CD11b was necessarily omitted because the FL1 channel was occupied by GFP fluorescence. Essentially all CD45dim cells coexpress CD11b, and relative levels of CD45 can distinguish microglia (CD45dim) from leukocytes (CD45high).25Ford AL Goodsall AL Hickey WF Sedgwick JD Normal adult ramified microglia separated from other central-nervous-system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex-vivo antigen presentation to myelin basic protein-reactive Cd4(+) T-cells compared.J Immunol. 1995; 154: 4309-4321PubMed Google Scholar, 26Sedgwick JD Schwender S Imrich H Dorries R Butcher GW Termeulen V Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system.Proc Natl Acad Sci USA. 1991; 88: 7438-7442Crossref PubMed Scopus (586) Google Scholar, 27Renno T Krakowski M Piccirillo C Lin JY Owens T TNF-α expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis. Regulation by Th1 cytokines.J Immunol. 1995; 154: 944-953PubMed Google Scholar Cells were then washed once in RPMI 1640 medium with 10% FBS and twice in annexin V-binding buffer (10 mmol/L HEPES, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2, pH 7.4) before incubation for 15 minutes at room temperature in a mixture of APC-conjugated annexin V (BD Biosciences), which labels apoptotic cells, and the viability marker 7-amino-actinomycin D (7-AAD), which allows exclusion of dead cells (BD Biosciences). For BrdU analysis, cells from chimeric and nonchimeric mice were washed in staining buffer (Hanks' balanced salt solution containing 2% FBS and 0.1% sodium azide) and for surface antigen labeling incubated with PE-conjugated anti-CD45 and PerCP-Cy5.5-conjugated anti-CD11b antibodies (BD Biosciences). Cells were then washed in staining buffer and fixed, permeabilized, and stained with APC-conjugated anti-BrdU antibody for intracellular BrdU detection according to instructions provided with the APC BrdU flow kit (BD Biosciences). All stainings were analyzed on a BD FACSCalibur flow cytometer and BD CellQuest Pro software (BD Biosciences, San Jose, CA). Isotype, antigen omission (for BrdU), and autofluorescence (for GFP, 7-AAD, and annexin V) controls served to determine fluorescence levels for positive staining. Specificity of annexin V binding was additionally tested by blocking with recombinant annexin V. For quantification of flow cytometry data, cells were gated on side scatter (SSC) versus CD11b and SSC versus either intermediate expression of CD45 to identify parenchymal microglia (CD45dimCD11b+), or high levels of CD45 to identify macrophages (CD45highCD11b+). Note that identification of apoptotic microglia in chimeric mice was based solely on SSC versus CD45dim expression for reasons outlined above. All microglia/macrophages included in annexin V analyses were additionally gated to show only viable cells, by excluding late apoptotic and necrotic cells positive for 7-AAD from the analysis. Note that certain flow cytometry profiles included in figures are shown for descriptive purposes only, and may have alternate gating strategies; this information is specified in the figure legends. Numbers of microglia or macrophages in one hippocampus were calculated as the number of gated CD45dimCD11b+ microglia or gated CD45highCD11b+ macrophages multiplied by the reciprocal fraction of the sample volume used and multiplied by two. This last step was taken because each hippocampal sample was split in two, ie, half was used for annexin V analysis and the other half for BrdU incorporation. For C57BL/6 mice, the data are presented as the average of these duplicates. For chimeric mice, the data are presented from our analysis of BrdU-stained cells because both CD45 and CD11b antibodies could only be included there. Note that number data generated from groups of B6(CD45.1) and C57BL/6 mice used for comparison with chimeric mice were also estimated using this approach. Numbers of microglial and macrophage subpopulations were calculated by applying the proportion of the subset to the total number estimate. Preparation and staining of hippocampal homogenate inevitably leads to cells loss, and the cell numbers and proportions presented are based on cells remaining in suspension. For stereological quantification of total microglial numbers in the dentate gyrus, vibratome sections were processed for immunohistochemical visualization of CD11b+ microglia. PP-lesioned mice were sacrificed under pentobarbital anesthesia by exsanguination and intracardiac perfusion with 5 ml of Sørensen's phosphate buffer (25 mmol/L KH2PO4 and 125 mmol/L Na2HPO4, pH 7.4) followed by 20 ml of Sørensen's phosphate buffer containing 4% paraformaldehyde. The brains were additionally fixed 1 hour in Sørensen's phosphate buffer containing 4% paraformaldehyde on ice and 21 hours in Sørensen's phosphate buffer with 1% paraformaldehyde at 4°C. Sections of 70-μm thickness were cut on a VT1000S vibratome (Leica, Nussloch, Germany) and transferred to de Olmos cryoprotectant solution [10 g of polyvinylpyrrolidone (Sigma-Aldrich, Brondby, Denmark) and 300 g of sucrose diluted in a mixture of 300 ml of ethyleneglycol (Merck, Glostrup, Denmark) and 700 ml of NaPO4 buffer] for long-term storage. Sections selected for CD11b labeling were rinsed in Tris-buffered saline (TBS; 50 mmol/L Trisma base and 110 mmol/L NaCl, pH 7.4), endogenous peroxidase activity was blocked in methanol containing 0.6% H2O2 for 10 minutes at room temperature, and sections were incubated in staining buffer (TBS containing 1% Triton X-100 and 10% FBS) for 1 hour at room temperature. Incubation with the anti-CD11b antibody (Serotec, Hamar, Norway) in staining buffer was done overnight at 4°C. Subsequent incubations with secondary goat anti-rat biotinylated antibody (Amersham Biosciences, Little Chalfont, UK) and horseradish peroxidase conjugated streptavidin in staining buffer were performed at room temperature for 1 hour. Labeling was visualized with 3,3′-diaminobenzidine, and sections were mounted on gelatinized microscope slides, dried overnight, stained with toluidine blue, dehydrated in graded ethanol, cleared in xylene, and coverslipped in Depex mounting medium. Visualization of apoptotic CD11b+ microglia expressing activated caspase-3 was done in 16-μm-thick cryostat sections mounted on microscope slides. Tissue sections were rinsed in TBS and in TBS containing 1% Triton X-100 (TBS-T) before incubation in staining buffer for 1 hour at room temperature. The tissue was subsequently incubated with rat anti-mouse CD11b antibody (Serotec) and rabbit anti-human activated caspase-3 antibody (Cell Signaling Technology, Boston, MA) diluted in staining buffer overnight at 4°C. Afterward the tissue was rinsed in TBS-T and incubated with Alexa Fluor 568-conjugated goat anti-rat IgG antibody (Invitrogen) and Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Invitrogen). The tissue was finally rinsed in TBS and stained with 4′,6-diamidino-2-phenylindole (DAPI). After a final rinse in TBS, sections were coverslipped in Prolong Gold anti-fade medium (Invitrogen). Visualization of activated caspase-3-positive cells in the free-floating vibratome sections was done as described for CD11b using 3,3′-diaminobenzidine as chromogen. Fragmented DNA in apoptotic microglia was visualized applying terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) together with CD11b and DAPI staining. Free-floating 70-μm-thick vibratome sections were rinsed in TBS and TBS-T before preincubation in staining buffer at room temperature and incubation with CD11b antibody (Serotec) at 4°C overnight. Sections were then rinsed in TBS-T and incubated for 1 hour at room temperature with an Alexa Fluor 568-conjugated goat anti-rat IgG antibody. After rinsing in TBS-T fluorescein isothiocyanate-conjugated dUTP nick end labeling was performed according to kit instructions (Roche Diagnostics, Hvidovre, Denmark). Finally, sections were rinsed in PBS, mounted on gelatinized microscope slides, dried, stained with DAPI, and
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