Ablation of TNF-RI/RII Expression in Alzheimer's Disease Mice Leads to an Unexpected Enhancement of Pathology
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.07.001
ISSN1525-2191
AutoresSara Montgomery, Michael A. Mastrangelo, Diala Habib, Wade C. Narrow, Sara A. Knowlden, Terry W. Wright, William J. Bowers,
Tópico(s)Immune Response and Inflammation
ResumoAlzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe memory loss and cognitive impairment. Neuroinflammation, including the extensive production of pro-inflammatory molecules and the activation of microglia, has been implicated in the disease process. Tumor necrosis factor (TNF)-α, a prototypic pro-inflammatory cytokine, is elevated in AD, is neurotoxic, and colocalizes with amyloid plaques in AD animal models and human brains. We previously demonstrated that the expression of TNF-α is increased in AD mice at ages preceding the development of hallmark amyloid and tau pathological features and that long-term expression of this cytokine in these mice leads to marked neuronal death. Such observations suggest that TNF-α signaling promotes AD pathogenesis and that therapeutics suppressing this cytokine's activity may be beneficial. To dissect TNF-α receptor signaling requirements in AD, we generated triple-transgenic AD mice (3xTg-AD) lacking both TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII), 3xTg-ADxTNF-RI/RII knock out, the cognate receptors of TNF-α. These mice exhibit enhanced amyloid and tau-related pathological features by the age of 15 months, in stark contrast to age-matched 3xTg-AD counterparts. Moreover, 3xTg-ADxTNF-RI/RII knock out–derived primary microglia reveal reduced amyloid-β phagocytic marker expression and phagocytosis activity, indicating that intact TNF-α receptor signaling is critical for microglial-mediated uptake of extracellular amyloid-β peptide pools. Overall, our results demonstrate that globally ablated TNF receptor signaling exacerbates pathogenesis and argues against long-term use of pan-anti-TNF-α inhibitors for the treatment of AD. Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe memory loss and cognitive impairment. Neuroinflammation, including the extensive production of pro-inflammatory molecules and the activation of microglia, has been implicated in the disease process. Tumor necrosis factor (TNF)-α, a prototypic pro-inflammatory cytokine, is elevated in AD, is neurotoxic, and colocalizes with amyloid plaques in AD animal models and human brains. We previously demonstrated that the expression of TNF-α is increased in AD mice at ages preceding the development of hallmark amyloid and tau pathological features and that long-term expression of this cytokine in these mice leads to marked neuronal death. Such observations suggest that TNF-α signaling promotes AD pathogenesis and that therapeutics suppressing this cytokine's activity may be beneficial. To dissect TNF-α receptor signaling requirements in AD, we generated triple-transgenic AD mice (3xTg-AD) lacking both TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII), 3xTg-ADxTNF-RI/RII knock out, the cognate receptors of TNF-α. These mice exhibit enhanced amyloid and tau-related pathological features by the age of 15 months, in stark contrast to age-matched 3xTg-AD counterparts. Moreover, 3xTg-ADxTNF-RI/RII knock out–derived primary microglia reveal reduced amyloid-β phagocytic marker expression and phagocytosis activity, indicating that intact TNF-α receptor signaling is critical for microglial-mediated uptake of extracellular amyloid-β peptide pools. Overall, our results demonstrate that globally ablated TNF receptor signaling exacerbates pathogenesis and argues against long-term use of pan-anti-TNF-α inhibitors for the treatment of AD. The inflammatory responses associated with Alzheimer's disease (AD) and their contributions to the course of the disease and resultant neurodegeneration are becoming better appreciated.1Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. 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Restraining tumor necrosis factor-alpha by thalidomide prevents the amyloid beta-induced impairment of recognition memory in mice.Behav Brain Res. 2008; 189: 100-106Crossref PubMed Scopus (87) Google Scholar Our laboratory previously demonstrated a pre-pathological up-regulation of TNF-α and correlating enhancement of F4/80-positive microglia/macrophage numbers in the 6-month-old triple-transgenic AD (3xTg-AD) mouse model that exhibits an age-related development of amyloid and tau pathological features and deficits in synaptic plasticity, including hippocampal long-term potentiation (LTP), reminiscent of human AD.15Oddo S. Caccamo A. Kitazawa M. Tseng B.P. LaFerla F.M. Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease.Neurobiol Aging. 2003; 24: 1063-1070Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar, 16Janelsins M.C. Mastrangelo M.A. Oddo S. LaFerla F.M. Federoff H.J. Bowers W.J. 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In further support of a potential contributory role of TNF-α to AD pathogenesis, long-term TNF-α overexpression via viral vector-based gene transfer leads to enhanced inflammation and marked neuronal cell death in this mouse model of AD.18Janelsins M.C. Mastrangelo M.A. Park K.M. Sudol K.L. Narrow W.C. Oddo S. LaFerla F.M. Callahan L.M. Federoff H.J. Bowers W.J. Chronic neuron-specific tumor necrosis factor-alpha expression enhances the local inflammatory environment ultimately leading to neuronal death in 3xTg-AD mice.Am J Pathol. 2008; 173: 1768-1782Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar TNF-α belongs to the TNF superfamily of ligands and promotes inflammatory signaling by coordinating innate responses. Both biologically active transmembrane TNF-α and soluble TNF-α are produced by microglia, astrocytes, and specific subpopulations of neurons.19Strohmeyer R. Rogers J. Molecular and cellular mediators of Alzheimer's disease inflammation.J Alzheimers Dis. 2001; 3: 131-157PubMed Google Scholar, 20Hanisch U.K. Microglia as a source and target of cytokines.Glia. 2002; 40: 140-155Crossref PubMed Scopus (1289) Google Scholar, 21Chung C.Y. Seo H. Sonntag K.C. Brooks A. Lin L. Isacson O. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in their vulnerability and protection.Hum Mol Genet. 2005; 14: 1709-1725Crossref PubMed Scopus (281) Google Scholar TNF-α signals through two distinct membrane glycoprotein receptors: TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII). Most cell types express TNF-RI, and either soluble TNF-α or transmembrane TNF-α is able to initiate signaling through this cognate receptor, whereas TNF-RII is primarily engaged by transmembrane TNF-α and is expressed by microglia and endothelial cells.22McCoy M.K. Tansey M.G. 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TNF-alpha inhibition as a treatment strategy for neurodegenerative disorders: new drug candidates and targets.Curr Alzheimer Res. 2007; 4: 378-385Crossref PubMed Scopus (177) Google Scholar A prospective open-label pilot study27Tobinick E. Gross H. Weinberger A. Cohen H. TNF-alpha modulation for treatment of Alzheimer's disease: a 6-month pilot study.MedGenMed. 2006; 8: 25PubMed Google Scholar was conducted on 15 patients with AD who were administered perispinal etanercept, a potent TNF-α antagonist, semiweekly; these patients claimed cognitive improvements in three independent tests, whereas untreated patients exhibited progressive cognitive decline. Such findings are promising, yet they undoubtedly spur debate regarding the safety and efficacy of TNF-α inhibition over the lifetime of an AD-afflicted individual. To better understand the effect of TNF-α signaling ablation during a protracted period in the context of progressive AD-related pathogenesis, we generated 3xTg-AD mice devoid of cognate TNF-RI and TNF-RII (3xTg-ADxTNF-RI/RII knock out [KO]). Herein, we demonstrate that 3xTg-ADxTNF-RI/RII KO mice exhibit higher amyloid and tau-related pathological burden at the age of 15 months than age-matched 3xTg-AD mice. Moreover, microglia in 3xTg-ADxTNF-RI/RII KO mice appear nonresponsive to ongoing development of AD pathological features in vivo and exhibit reduced amyloid-β (Aβ)42 phagocytosis activity in vitro. In aggregate, these data imply that long-term inhibition of TNF-α in the central nervous system without consideration of cell type–specific requirements for intact TNF-α signaling may result in dire consequences by accelerating AD-related pathological features and may ultimately lead to enhanced neurodegeneration. Triple-transgenic AD (3xTg-AD) B1 line and non-transgenic (Non-Tg) mice were previously generated.15Oddo S. Caccamo A. Kitazawa M. Tseng B.P. LaFerla F.M. Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease.Neurobiol Aging. 2003; 24: 1063-1070Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar, 28Oddo S. Caccamo A. Shepherd J.D. Murphy M.P. Golde T.E. Kayed R. Metherate R. Mattson M.P. Akbari Y. LaFerla F.M. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction.Neuron. 2003; 39: 409-421Abstract Full Text Full Text PDF PubMed Scopus (3155) Google Scholar The TNF-RI/RII KO mice were previously described.29Pryhuber G.S. Huyck H.L. Bhagwat S. O'Reilly M.A. Finkelstein J.N. Gigliotti F. Wright T.W. Parenchymal cell TNF receptors contribute to inflammatory cell recruitment and respiratory failure in Pneumocystis carinii-induced pneumonia.J Immunol. 2008; 181: 1409-1419Crossref PubMed Scopus (15) Google Scholar At the time of mouse crossing for this study, the C57BL/6 genetic background was the predominant background for our 3xTg-AD colony (six backcrosses), with the TNF-RI/RII line being completely on the C57BL/6 genetic background. A monogamous mating strategy was used to generate 3xTg-ADxTNF-RI/RII KO mice by crossing the 3xTg-AD and TNF-RI/RII KO mice until all genes were homozygous. Briefly, in the parental (P) generation, 3xTg-AD mice and C57BL/6 TNF-RI/RII knockout mice generated (F1) offspring composed of heterozygous 3xTg-AD and TNF-α- receptor genes [3xTg-AD+/− (TNF-RI+/−) TNF-RII+/−]. The F1 generation mice were backcrossed with 3xTg-AD mice to yield homozygous PS1, APPswe, and taup301L genes, resulting in an F2 generation designated as 3xTg-AD+/+ (TNF-RI+/−) TNF-RII+/−. Mice harboring homozygous 3xTg-AD and TNF-RI genes were generated by crossing F2 generation mice with each other to yield F3 mice [3xTg-AD+/+ (TNF-RI−/−) TNF-RII+/−]. Subsequently, F3 generation mice were crossed to generate mice homozygous for the 3xTg-AD, TNF-RI, and TNF-RII genes (Figure 1A). 3xTg-AD and 3xTg-ADxTNF-RI/RII KO mice were monogamously mated to produce offspring, which were housed until sacrificed at the indicated points. Age-matched 2-, 3-, 6-, 9-, 12-, and 15-month-old male mice were used in the immunohistochemical (IHC)/semiquantitative studies (n = 3 to 7 per experimental group). P1 pups were used to establish primary microglial cultures for phagocytosis analyses (N = 8 per genotype). For IHC analyses, mice were euthanized with an overdose of pentobarbital, followed by transcardiac perfusion with heparinized saline, then by 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB). Brains were extracted and postfixed overnight in 4% paraformaldehyde in 0.1 mol/L PB, then equilibrated with 20% sucrose in 0.1 mol/L PBS and transferred into 30% sucrose in 0.1 mol/L PBS. Brains were coronally divided into sections (30-μm thick) on a freezing stage-sliding microtome (Microtome, Walldorf, Germany) and stored at −20°C in cryoprotectant until use for IHC analysis. All mice were housed and bred in accordance with the University of Rochester (Rochester, NY) requirements for animal welfare and care. Mice were on a 12-hour light-dark cycle and were allowed food and water ad libitum. Genotyping of transgenic mice was conducted via conventional PCR using mouse genomic DNA extracted from tail biopsy specimens. PCR was performed in 25-μL reaction mixtures, each containing 100 ng/μL genomic DNA, 20 μmol/L of each primer, 10 mmol/L deoxyribonucleotide triphosphate mixture, 2.5 μL of 10× Taq buffer, and 0.3 μL of Taq polymerase (Promega, Madison, WI). The primers specific for the APP transgene amplification used were as follows: 5′-GCTTGCACCAGTTCTGGATGG-3′ (forward) and 5′-GAGGTATTCAGTCATGTGCT-3′ (reverse). The following conditions were used: 5 minutes at 94°C; 30 seconds at 94°C, 30 seconds at 53°C, and 1 minute at 72°C for 20 cycles; and then 1 minute at 72°C using a MyCyclerThermal Cycler (BioRad, Hercules, CA). The tau-specific primers used were as follows: 5′-GAGGTATTCAGTCATGTGCT-3′ (forward) and 5′-TTCAAAGTTCACCTGATAGT-3′ (reverse). The following conditions were used: 94°C for 5 minutes; 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute for 25 cycles; and then 72°C for 3 minutes using the MyCyclerThermal Cycler. For PS1, the PCR was doubled with forward primer, 5′-CACACGCAACTCTGACATGCACAGGC-3′, and reverse primer, 5′-AGGCAGGAAGATCACGTGTTCAAGTAC-3′, at 94°C for 2.5 minutes; 94°C for 40 seconds, 62°C for 40 seconds, and 72°C for 1 minute for 35 cycles; and then 72°C for 3 minutes using an Eppendorf Mastercycler Gradient system (Eppendorf, Hauppauge, NY). Subsequently, half of the PCR product was digested with BstEII at 60°C for 1 hour because the primers also amplify the endogenous PS1 gene. Two bands, 300 and 250 bp, indicated homozygous mice. TNF-RI amplification was performed using three primers with the following oligonucleotide sequences: 5′-TGTGAAAAGGGCACCTTTACGGC-3′, 5′-GGCTGCAGTCCACGCACTGG-3′, and 5′-ATTCGCCAATGACAAGACGCTGG-3′. The amplification conditions used were as follows: 94°C for 3 minutes; 94°C for 20 seconds, 64°C for 30 seconds with −0.5°C per cycle, and 72°C for 35 seconds for 12 cycles; 94°C for 20 seconds, 58°C for 30 seconds, and 72°C for 35 seconds for 25 cycles; and then 72°C for 2 minutes using a MyCyclerThermal Cycler to amplify a 300-bp band for homozygous mice. Similarly, TNF-RII cDNA was amplified using the following primer sequences: 5′-CCTCTCATGCTGTCCCGGAAT-3′, 5′-AGCTCCAGGCACAAGGGCGGG-3′, 5′-GCCCTGAATGAACTGCAGGACG-3′, and 5′-CACGGGTAGCCAACGCTATGTC-3′. The conditions were as follows: 94°C for 3 minutes; 94°C for 1 minute, 62°C for 1 minute, and 72°C for 1 minute for 32 cycles; and then 72°C for 10 minutes using MyCyclerThermal Cycler. Homozygous mice were indicated by a 400-bp amplification product. PCR products were separated electophoretically on 0.5% agarose gels and visualized via ethidium bromide. Brain sections were washed with 0.15 mol/L PB for 2 hours to remove cryoprotectant, subsequently mounted on SuperFrost Plus slides (VWR International, West Chester, PA), and allowed to dry. The slides were hydrated in dH-2O for 5 minutes before staining with 0.02% Cresyl violet acetate in 0.25% acetic acid for 30 minutes. To destain, sections were rinsed in three changes of dH-2O, then placed in 50% ethanol for 1 minute, followed by 70% ethanol for 1 minute. Sections were then completely dried, dipped in xylene, and coverslipped. Sections were analyzed via the MCID 6.0 ELITE Imaging Software Program (Interfocus Imaging, Cambridge, UK) using the length tool to measure (in pixels) five regions of the hippocampus, including the Cornu Ammonis (CA)1, CA2, CA3, dentate gyrus (DG), and lacunosum molecular layer under ×40 magnification. Measurements were taken at specific bregma positions, and 4–51 values were used per bregma position per anatomical region. Identical measurements were performed for a particular bregma position and brain region between 3xTg-AD and 3xTg-ADxTNF-RI/RII KO mice. Measurements were analyzed with a two-way analysis of variance and a Bonferroni's multiple comparisons posttest (N = 3 to 7 per genotype per time point). 3xTg-AD and 3xTg-ADxTNF-RI/RII KO mice at the age of 2 months were stereotactically infused with 2 μL (3 × 109 transducing units) of recombinant adeno-associated virus serotype-2 (rAAV2)–TNF-α and rAAV2–enhanced green fluorescence protein (eGFP), as previously described.18Janelsins M.C. Mastrangelo M.A. Park K.M. Sudol K.L. Narrow W.C. Oddo S. LaFerla F.M. Callahan L.M. Federoff H.J. Bowers W.J. Chronic neuron-specific tumor necrosis factor-alpha expression enhances the local inflammatory environment ultimately leading to neuronal death in 3xTg-AD mice.Am J Pathol. 2008; 173: 1768-1782Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar The following antibodies were used to IHC stain 3xTg-AD and 3xTg-AD × TNF-RI/RII KO brain sections at the indicated dilutions: microglia/macrophage-specific cell marker [anti-ionized calcium-binding adaptor molecule 1 (Iba1), rabbit polyclonal, 1:750; Wako, Richmond, VA]; astrocyte-specific cell marker [polyclonal rabbit anti-glial fibrillary acidic protein (GFAP), 1:1000; Dako, Richmond, VA]; anti-Aβ 1–42 clone 12F4 reactive to the C-terminus of β-amyloid and specific for the isoform ending at amino acid 42 (1:1000; Signet Labs, Berkeley, CA); anti-human phosphorylated-tau monoclonal AT-180 specific human tau recognizing doubly phosphorylated Thr231 and Ser235 residues (1:500; Pierce, Rockford, IL); anti-human tau HT7, specific to residues 159 to 163 (1:200; Pierce); anti-amyloid precursor protein A4, corresponding to the NPXY motif of hAPP (Clone Y188, 1:750; AbCam, Cambridge, MA); paired helical filament (PHF)-1 recognizing singly or doubly phosphorylated tau at Ser396 or Ser404 residues (1:30; provided by Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY); and anti-human tau HT7, reactive to residues 159 to 163 (1:200; Pierce). To characterize basal synaptic transmission and function, electrophysiological procedures were performed on 6- to 7-month-old 3xTg-AD, 3xTg-AD × TNF-RI/RII KO, and Non-Tg mice under deep urethane anesthesia (1.5 g/kg i.p., administered in one dose and supplemented with 0.1 to 0.2 g/kg as required before the onset of data collection). All mice were placed in a stereotaxic apparatus, and body temperature was maintained between 36°C and 37°C by using an electrical heating blanket. An incision was made to expose the skull surface, and small skull holes were drilled above the CA3 region (anterior-posterior [AP], -2.20; medial-lateral [ML], -2.5; ventral-dorsal [VD], -2.3 to −2.5 mm) and the contralateral CA1 area (AP, -2.20; ML, 1.50; VD, -1.6 mm). Skull holes for ground and reference electrodes (jewelry screws attached to miniature connectors) were made in the bone overlying the prefrontal cortex and cerebellum, respectively. All stereotaxic measurements were based on the anatomical work of Paxinos and Franklin.30Paxinos G. Franklin K.B.J. The Mouse Brain Stereotaxic Coordinates.in: 2nd ed. Academic Press, San Diego2001: 296Google Scholar Final ventral placements of the CA3 stimulation and the CA1 recording electrodes were adjusted to elicit the maximal amplitude of field excitatory post-synaptic potentials (fEPSPs) and paired-pulse facilitation (100-millisecond interstimulus interval) in area CA1 in response to contralateral CA3 stimulation. Stimulation of CA3 (0.2-millisecond pulses every 30 seconds, intensity adjusted to yield 50% to 60% of maximal fEPSP amplitude) was provided by a concentric bipolar electrode (Rhodes Medical Instruments Series 100; David Kopf, Tujunga, CA) connected to a stimulus isolation unit providing a constant current output (PowerLab/16-second system with ML 180 Stimulus Isolator; AD Instruments, Toronto, ON, Canada). All fEPSPs in stratum radiatum of CA1 were differentially recorded (Teflon-insulated stainless steel wire, 125-μm tip diameter) against a reference electrode placed in the bone overlaying the cerebellum. The CA1 signals were amplified, filtered (0.3 to 1 kHz), digitized (10 kHz), and stored for subsequent off-line analysis using PowerLab system running Scope software, version 4.0.2 (AD Instruments, Toronto, Canada). Before formal data collection, input-output curves were established for each animal by recording fEPSPs in CA1 in response to CA3 stimulation between 0 and 400 μA (50-μA increments). Based on these input-output curves, a stimulation intensity eliciting between 50% and 60% of the maximal fEPSP amplitude was chosen for the subsequent experiment. For each mouse, 60 initial baseline fEPSPs (every 30 seconds) were recorded. After stable baseline recordings, all animals received a burst of high-frequency stimulation (HFS) to the CA3 area (100 pulses at 100 Hz). Recordings of fEPSPs in CA1 (every 30 seconds, as in baseline) in response to single-pulse CA3 stimulation continued for 1 hour after the HFS episode. At the end of each experiment, mice were intracardially perfused with 10% paraformaldehyde, their brains were extracted, and standard histological techniques were used to verify all electrode placements. Data obtained with inaccurate placements were excluded from the data analysis. All electrophysiological data are expressed as mean ± SEM. The maximal fEPSP amplitude was analyzed offline by the Scope software. Subsequently, amplitude data were averaged over 10-minute intervals, and these averages were normalized by dividing all data for each mouse by the average baseline (pre-HFS) amplitude of that animal. All LTP data were analyzed using analyses of variance and, where statistically appropriate, simple effects and Tukey's post hoc tests, all of which were computed using GraphPad Prism software (GraphPad, La Jolla, CA). Brain sections were washed with 0.15 mol/L PB to remove cryoprotectant, followed by a 20-minute incubation with 3% H2O2 in 0.15 mol/L PB to quench endogenous peroxidase activity. Epitope retrieval was used for Aβ peptide-specific stains using 70% formic acid for 15 minutes. IHC processing was performed as previously described.31Mastrangelo M.A. Bowers W.J. Detailed immunohistochemical characterization of temporal and spatial progression of Alzheimer's disease-related pathologies in male triple-transgenic mice.BMC Neurosci. 2008; 9: 81Crossref PubMed Scopus (174) Google Scholar Slides were visualized, and staining intensities were quantified. Statistics for the IHC experiments were performed using a two-way analysis of variance and a Bonferroni's multiple comparison test via GraphPad Prism software. By using an Olympus AX-70 microscope equipped with a motorized stage (Olympus, Center Valley, PA) and MCID 6.0 ELITE Imaging Software Program (Interfocus Imaging subsidiary of GE Healthcare, Cambridge, England), the hippocampal CA1 region of immunohistologically stained sections was quantified under ×20 magnification in a blinded fashion. Each image represented one-twelfth of the total hippocampus. Ten to fifty images per mouse were analyzed and averaged for statistical analysis. N = 3 to 7 was used per time point per genotype. Photomicrographic images were processed consistently where brightness and contrast alterations were applied identically over all images within an experimental data set using Photoshop CS3 (Adobe Systems, Inc., San Jose, CA). No other image processing changes were applied. Microglia were derived from 3xTg-AD or 3xTg-ADxTNF-RI/RII KO postnatal day 1 (P1) pups. Cerebral cortexes were isolated, and meninges were removed and minced in Hanks' balanced salt solution (HBSS; Invitrogen, Frederick, MD). Cells were dissociated by trituration in minimum essential media (Invitrogen, Frederick) containing Earle's salts, l-glutamine, 0.01% pyruvate, 0.6% glucose, 4% fetal bovine serum, and 6% horse serum (complete medium); centrifuged; an
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