Population Control of Resident and Immigrant Microglia by Mitosis and Apoptosis

【摘要】  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 Using bone marrow chimeric mice, we found that the lesion-induced proliferative capacity of resident microglia superseded that of immigrant microglia, whereas lesion-induced kinetics of apoptosis were comparable.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.
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Microglia represent the first line of defense against pathogens or injury in the central nervous system (CNS).1 By dynamically surveying the CNS, microglia serve important maintenance functions for neurons, with which they have intimate contact.2,3 Key functions include metabolism of nucleosides and purines,2,3 phagocytosis, and production of growth factors and cytokines.4,5 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,6 develop symptoms of presenile dementia.7
Microglia are unique cells in the CNS parenchyma, being innate immune cells and having a mesodermal origin. In line with their myeloid lineage,8 slow microglial turnover in normal CNS9 and enhanced recruitment during reactive microgliosis10,11 indicate that microglia are potentially replaceable cells, making microglia and their myeloid progenitors promising candidates for active cellular therapy in CNS disease and injury.12,13 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,11 proliferation,14 or migration from juxtaposed regions.15 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.16 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.

【关键词】  population resident immigrant microglia apoptosis

Materials and Methods

Animals

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)17 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.

PP Lesion

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,18-21 performed as previously described.11 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 C3 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.

BM Transplantation

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.10,11 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.11 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.

Real-Time Polymerase Chain Reaction (PCR)-Based Testing for UbC-GFP Zygosity

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 1x 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- (LT-) 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.

Real-Time PCR-Based mRNA Analysis

RNA was extracted using RNeasy Protect mini kits (Qiagen, Hilden, Germany) or TRIzol (Invitrogen, Taastrup, Denmark), as previously described for PP-lesioned hippocampus samples.22,23 Complementary DNA was synthesized from 0.4 µg of total RNA by reverse transcription, using previously established conditions.23 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 1x, 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.23 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.

Labeling of Proliferating Cells in Vivo

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.

Flow Cytometry

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.24 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-FcIII/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.11,22

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).25-27 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.

Histology

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 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 coverslipped in Prolong Gold anti-fade medium.

For confocal analysis of microglial clusters, free-floating 70-µm-thick vibratome sections were rinsed in TBS and TBS-T before preincubation in staining buffer at room temperature. The tissue was then incubated with rat anti-mouse CD11b antibody (Serotec) overnight at 4??C. Sections were rinsed in TBS-T and incubated for 1 hour at room temperature with Alexa Fluor 488 conjugated goat anti-rat IgG antibody. Sections were then rinsed in TBS and stained with propidium iodide. After a final rinse in TBS, the sections were mounted in TBS and coverslipped using Gerbatol mounting medium. Fluorescence stainings were analyzed using a Leica TCS 4D/DM IRB laser-scanning confocal microscope.

For visualization of mitotic microglia, 16-µm-thick cryostat sections were rinsed in TBS and in TBS-T before immersion in 1 mol/L HCl for 10 minutes on ice, 2 mol/L HCl for 10 minutes at room temperature, and 2 mol/L HCl for 20 minutes at 37??C. Tissue was rinsed in TBS-T and incubated with staining buffer for 1 hour at room temperature followed by incubation with biotinylated tomato lectin (Sigma, Copenhagen, Denmark) and rat anti-BrdU antibody (Abcam, Cambridge, UK) diluted in staining buffer at 4??C overnight. The tissue was then rinsed in TBS-T and incubated with Alexa Fluor 568-conjugated goat anti-rat IgG antibody and Alexa Fluor 488-conjugated streptavidin. The tissue was finally rinsed in TBS and stained with DAPI, and sections were coverslipped in Prolong Gold anti-fade medium.

In situ visualization of GFP+ BM-derived microglia was done in 16-µm-thick cryostat sections. Sections were rinsed in TBS before preincubation in TBS containing 10% FBS for 1 hour. The tissue was incubated overnight with rat anti-mouse CD11b antibody (Serotec) in TBS containing 10% FBS and 0.5% Triton X-100. The sections were subsequently rinsed in TBS and incubated with an Alexa Fluor 568-conjugated rabbit anti-rat IgG antibody for 1 hour, rinsed again in TBS, and coverslipped in Prolong Gold anti-fade medium with DAPI. Fluorescence stainings were analyzed using an Olympus BX51 fluorescence microscope and DP70 digital color camera.

Stereology

Histological quantification of microglia was based on the protocol for counting neurons in the hippocampus, by applying the optical fractionator28 adapted for unbiased counting of microglia in the dentate gyrus as previously described.29 All counting was performed using the computer-assisted stereological toolbox (CAST)-Grid software (Visiopharm, Hørsholm, Denmark) connected to an Olympus BX50 microscope equipped with motorized stage and focus units. Each brain was sectioned horizontally in four parallel series of sections. One was used for stereological counting giving a section sampling fraction ssf of 1/4. Cells were sampled in counting frames of 644 to 988 µm2 . The area sampling fraction asf was calculated as a(frame)/a(step). The thickness sampling fraction tsf was calculated as the height of the optical disector probe h (8 or 10 µm) divided by the average height of the sections t (tsf = h/t). Total cell number N was estimated using the equation: N = QC ?

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作者单位：From the Medical Biotechnology Center* and the Departments of Anatomy and Neurobiology and Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark; the Department of Medical Microbiology and Immunology,¶ University of Aarhus, Aarhus, Denmark; a