The generation of CX3CR1 deficient mice and TgCRND8 mice has been previously described. Briefly, the CX3CR1 gene locus underwent targeted deletion and direct replacement by a green fluorescent protein (GFP) reporter gene. CX3CR1+/− mice previously backcrossed to C57BL/6 mice for more than 10 generations were crossbred with TgCRND8 mice to obtain CRND8/CX3CR1−/−, CRND8/CX3CR1+/− and CRND8/CX3CR1+/+ mice. Experiments in which we quantified amyloid plaque density, Aβ concentrations and APP processing/cleavage were done in male mice. All other experiments including quantification of Aβ within microglia and in vivo imaging experiments were done in mixed gender mice but with equal gender distribution on each experimental group. Experimental protocols were approved by the Northwestern University Feinberg School of Medicine Institutional Animal Care and Use Committee.
In vivo imaging with two photon microscopy
GFP-labeled microglia and Methoxy-X04 (MX04) labeled plaques were imaged through a thinned skull preparation as previously described (Grutzendler et al., 2002
). Briefly, transgenic mice were anesthetized with Ketamine/Xylazine and the skull was exposed with a midline scalp incision. About a 1 mm diameter skull region over the somatosensory cortex was thinned with a high speed drill and a microsurgical blade to a final thickness of ~30μm. The skull was attached to a custom-made steel plate to stabilize the head while imaging. A mode-locked Ti-sapphire laser (Coherent Inc.) was used for two-photon excitation (Prairie technologies) and tuned to 835 nm for dual imaging of GFP and MX04 or 890 nm for GFP alone. Emission wavelengths for MX04 and GFP were separated from each other with a 490 nm dichroic. Images were taken using a water immersion objective (Olympus 40x or 60x, 0.8 and 0.9 N.A. respectively) at z-steps of 1–2 μm and zooms of 1–4x. In most cases images were taken at depths up to ~150 μm below the pial surface. The same regions were relocated at different time points using the unchanged pattern of pial blood vessels as reference as well as registration of X, Y, Z coordinates of each scanning area at the initial time point and relocation of the same coordinates using a precision motorized stage at subsequent time points. The precise identity of the areas is confirmed by the presence of unambiguous microvascular shadows or plaque patterns
In vivo subarachnoid Aβ42 delivery
A ~500 μm area of the skull was thinned with a high speed micro drill at specific cranial stereotaxic coordinates (3.0mm mediolateral, −8.0mm anterioposterior from Bregma). The tip of a 27g needle was used to make a small cut on the surface of the thinned skull area and into the underlying dura without touching the brain parenchyma. Custom-made polypropylene tubing with a final outer diameter of 90μm was inserted ~300 μm into the subarachnoid space and fixed to the skull with cyanoacrylate glue. The tube was connected to a programmable syringe pump through a Hamilton syringe and 10μl (0.4 μM) of fluorescently labeled Aβ42 monomers (HiLyte Fluor 555-Aβ42, AnaSpec, Inc) freshly dissolved in sterile aCSF immediately prior to injection was infused at a rate of 0.2 μl/min. After infusion, tubing was removed and animal scalp was sutured. Animals were imaged in vivo by TPM or in fixed tissue by confocal microscopy at various time intervals after infusion.
Quantification of infused fluor-Aβ42 phagocytosis
Analysis of microglia phagocytosis of fluor-Aβ42 and presence of Thioflavin-S positive material within microglia was performed using NIH ImageJ software on confocal images of brain sections obtained from Fluor-Aβ infused mice. All images were obtained with standardized parameters of laser intensity, gain and pinhole size. a) fluor-Aβ42 and thioflavin-S content within microglia and astrocytes: microglial or astrocyte cell bodies were identified in z-projections from confocal images. A region of interest (ROI) was drawn around the cell body perimeter as well as a mirror ROI control immediately outside of each microglia or astrocyte. Mean fluorescence intensity values were measured for each cell and control ROI in both red channel for Aβ42 (HiLyte-Fluor-555) and blue channel for Thioflavin-S positive fibrillar material. b) Quantification of Aβ fluorescence outside of microglia: custom made NIH Image J Macros were written with the purpose of masking and subtracting areas where microglia were located. Mean fluorescence intensity values were obtained from the resulting area after subtraction of microglia regions. For the purpose of our study any fluor-Aβ deposit that was not located inside microglia was considered to be in the extracellular space, given our finding of the limited intake of fluor-Aβ by astrocytes (supplementary data
). Ultimately however, it is not possible to determine unambiguously if deposits are extracellular given the very small size of this space which is beyond the resolution of light microscopy
Quantification of Thioflavin-S labeled Amyloid Plaques
Sagital sections of the entire left hemisphere were obtained with a cryostat at 50 μm thickness. 8 sections for each hemisphere were selected and visually matched between animals to represent the same brain regions. Sections from all genotypes were stained with thioflavin-S with a standardize concentration and time. Whole brain sections were imaged with a fluorescence microscope (Zeiss axiovert 200M, objective 20x, 0.75NA) using identical parameters of gain and exposure in the CCD camera. All images were stitched together to generate a single image of the entire brain slice. A custom-made thresholding macro based on NIH-Image J was used to detect all fluorescent amyloid plaques using exactly the same parameters for all sections and genotypes. NIH-Image J particle counting plug-in was used to calculate the total number of amyloid plaques per brain section. A subset of images was counted manually by an unbiased individual to corroborate the accuracy of our automated counting. The use of automated counting allowed us to count a much larger number of plaques representing the majority of the hemisphere than would have been possible with stereological methods.
Quantification of MXO4-labled plaque area in vivo
Mice were injected only once with MX04 2 days before the first imaging time point. TPM imaging was done as described above. Image stacks containing plaques were run through a “despeckle” filter to remove noise, and then a maximal projection through a consistent number of slices was taken using NIH image J software. To achieve accurate plaque area measurements regardless of minor brightness differences across time points, images were thresholded for each time point at the average pixel brightness value between a consistent plaque area and the adjacent background.
Quantification of 4G8 and A11 fluorescence inside microglia phagolysosomes
Cryosections (50μm thick) underwent sodium citrate antigen retrieval and were immunolabled with the following antibodies: 4G8 (Covance), A11 (Biosource Invitrogen), IBA1 (Wako Chemicals USA, Inc), and LAMP1 (Developmental Studies Hybridoma Bank (Iowa) clone 1D4B) according to manufacturer protocols. Confocal images of plaques selected for their similar size, location and cortical layer were obtained for quantitative imaging using an Olympus FV10i Confocal Microscope (60X objective N.A. 1.2). Image z-stacks were taken with standardized laser intensity, gain and pinhole sizes to allow inter-animal comparisons. LAMP1-positive lysosomes inside IBA1-labeled microglia have distinct fluorescence intensities in comparison to LAMP1-positive structures outside microglia. Using NIH Image J, we applied a fluorescence threshold to the LAMP1 signal and created a binary image that was then combined with the other fluorescent channels in the z-stack to measure 4G8, A11, and Thioflavin S fluorescence intensities within the microglial LAMP1 structures. Measurements were performed on each optical slice of a given z-stack and then averaged.
Microglia culture and in vitro phagocytosis assay
One day old CX3CR1+/+ and −/− mouse littermates were used for primary cultures of cerebral cortical cells. These cell cultures include abundant microglia, astrocytes and neurons as confirmed by immunocytochemistry. Briefly, following cell dissociation, a mixture of equal number of brain cells from both genotypes was adjusted to 106 cells/ml in DMEM/10% fetal bovine serum (FBS), followed by plating of the cell suspension on a culture flask. Fifty percent of the medium was replaced at days 3, 10 and 17. At day 21, cells reaching 90% confluence were trypsinized and re-seeded to 24-well plates containing poly-lysine coated coverslips at a density of 5×104 cells/ml. The re-seeded cells were rinsed in DMEM containing 10% FBS, pretreated with or without 10μg/ml CX3CR1 neutralizing antibody for 30min and then incubated for 2 hours in the same medium containing either 2μm diameter fluorescent microspheres or 0.05mg/ml fluorescently-conjugated Aβ (HiLyte Fluor 555- Aβ42 AnaSpec, Inc) in DMEM. After washing with DMEM, cells were fixed in 2% paraformaldelhyde/PBS for immunocytochemistry. Fixed cells were rinsed in PBS, blocked in 3% normal goat serum/0.3% triton X-100 for 30min and then incubated with primary antibodies (IBA1, rabbit polyclonal, 1:500 and GFP, mouse monoclonal, 1:1000) overnight. CX3CR1−/− microglia were differentiated from CX3CR1+/+ microglia because they had positive labeling with both IBA1 and GFP antibodies while CX3CR1+/+ microglia were only labeled by IBA1. We also excluded the possibility of other cell types expressing either IBA1 or GFP in control experiments in which we used dual labeling with antibodies specific for astrocytes (GFAP) or neurons (MAP2). In separate culture preparations containing only CX3CR1−/− microglia we determined that 100% of IBA1-positive microglia were GFP positive. Microglia of each genotype represent ~50% of the total microglia population in these mixed cell cultures. Minor differences in the proportion of microglia of each genotype do not affect the final results of the phagocytosis assay given that in a mixture of cells there is equal likelihood for each individual cell regardless of its genotype to be exposed to Fluor 555-Aβ42 or microspheres. For quantification of microglia uptake of microspheres or Aβ, we used a custom-made macro with NIH-Image J software for automated measurement of total fluorescence intensity within individual microglia.
Quantification of microglia, neuron and synapse density around plaques
Cryosections (50μm thick) were immunolabled with any of the following antibodies: Anti IBA1 (Wako Chemicals USA, Inc), Synaptophysin (Chemicon NeuN (Chemicon) according to manufacturer protocols. Confocal images of plaques selected for their similar size, location and cortical layer (layers III to VI in somatosensory cortex) were obtained for quantitative imaging using a Zeiss LSM 510 Meta UV microscope (63X objective N.A. 1.4). Images were taken at standardized laser intensity, gain and pinhole sizes to allow inter-animal comparisons. Quantification of microglia and NeuN postitive neuron number was performed using the Image J threshold/analyze particle features. Results were validated by manual counting in a subset of stacks. To control for possible differences in microglia density between CX3CR1 genotypes in the absence of AD pathology, we measured the density of microglia in non-AD/CX3CR1 mutant mice which did not differ between genotypes in the brain region analyzed. We have observed full colocalization of IBA1 and F4/80 (an additional microglia marker) demonstrating that all microglia are IBA1 immunoreactive. Furthermore, IBA1 has a 100% colocalization with GFP-labeled cells in the brain parenchyma of CX3CR1 knock in mice. Thus IBA1 labels all brain microglia but cannot differentiate them from infiltrating macrophages given that macrophages also label with IBA1. Microglia density around plaques was quantified in blindly selected plaques of the same average size to eliminate size as a variable which can lead to differences in microglia number unrelated to the CX3CR1 genotype..
For synaptophysin quantification, multi-channel 8 bit images were obtained in which plaques were labeled with Thioflavin S in one channel and synaptophysin in a second channel. Using a custom ImageJ macro, the resultant stacks were analyzed slice by slice to determine the fluorescence of synaptophysin as a function of distance from the plaque edge. The plaque edge was determined by thresholding the Thioflavin S channel at a constant predetermined value of 115. This edge was selected and then enlarged by 2 μm (the selection grows in all directions out from its center for 2 μm) to form an even starting point. Then, 10 donut-shaped selection bands of 3 μm width were created at 3 μm intervals, covering a 30 μm circular area out from the evened plaque edge. This essentially creates concentric circular bands of selection, which were then used to measure average fluorescence intensity in the synaptophysin channel, with the first band nearly adjacent to the plaque (all pixels 2–5 μm from the thresholded plaque edge), and the last band about 30 um away from the plaque edge (all pixels 32–35 μm from the thresholded plaque edge).
Quantification of microglia cell body and process dynamics in vivo
a) For cell body motility, in vivo TPM images of both microglia and plaques were taken over 4–7 days. For long-term monitoring, a subset of mice was imaged at 1 and 4 month intervals. Microglia cell bodies adjacent to the plaque were counted for each time point and microglia stability was calculated as the percent of all adjacent cell bodies in the first time point which remained in the same location relative to the plaque at the later time point. b) For cell process dynamics, image stacks of microglia processes at two time points 1 hour apart were aligned using ImageJ plugins, maximally projected, matched for brightness, and then thresholded. Each time point was subtracted from the other in order to determine regions which were “lost” (apparent in the first image but not the second) or “gained” (apparent in the second image but not the first). The areas of regions of gains or losses above an arbitrary cutoff of 3.2 μm2 were then measured and counted per image stack.
Microglia proliferation assay
Mice received daily injections of bromodeoxyuridine (BrdU) (50 mg/kg, i.p.) for 14 days. Brains were removed following 4% PFA perfusion and cryosectioned at 50μm thickness. BrdU was detected after denaturing with 5N HCl followed by incubation with rat anti-BrdU antibody (1:400, Serotec, Oxford, UK) and rabbit anti-IBA1 antibody for microglia detection. BrdU and IBA1 were detected with AlexaFluor 555/488 conjugated secondary antibodies and imaged with confocal microscopy. BrdU-labeled microglia were quantified from confocal image stacks in areas in the vicinity (25 μm) and distant from plaques.
Aβ42 and Aβ40 were measured by commercially available ELISA kits (Biosource). Brain hemispheres of CX3CR1+/+, CRND8/CX3CR1+/+ and CRND8/CX3CR1−/− mice were homogenized in cold 5 M guanidine HCl/50mM Tris HCl. ELISA assays were performed according to manufacturer’s protocol.
Western Blot for APP and C-terminal fragments
Brain tissue lysates were prepared in ice-cold TEEN-Tx (TEEN buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA and 150 mM NaCl) containing 1% Triton X-100) by sonication. This was followed by centrifugation, protein quantification, addition of buffer, and boiling for 5 minutes. Proteins were separated using 15% tris-tricine polyacrylamide gel and incubated with APP C-terminal-specific rabbit anti-APP-CT20 antibody (1:10000; Calbiochem) and mouse anti-α-tubulin (1:5,000; Upstate). Densitometric quantification of band intensity was performed using NIH ImageJ software.
Mouse presenilin-1 and beta-site APP-cleaving enzyme 1(BACE1) mRNA expression were detected by RT-PCR. Briefly, total RNA was extracted and cDNA was obtained by Reverse Transcription according to manufacturer’s instruction. The following pairs of primers were used: 5′-AGC ATG ACA GGC AGA GAC TTG ACA-3′ and 5′-AAC GTA GTC CAC GGC GAC ATT GTA-3′ for presenilin 1, 5′-ATC TAC ACG CCC TTC ACG GAG G-3′ and 5′-TGG GCA GTT TCC ACC AGC ATC-3′ for presenilin 2, 5′-AGG GCT ACT ATG TGG AGA TGA CCG TA-3′ and 5′-TCC CAC TGT CCA CAA TGC TCT TGT-3′ for BACE1, 5′-AGA TGA GAA TGC CAG TCG CTC CTT-3′ and 5′-TGC ACA GTT GAG GTT CCC GAC TAA-3′ for BACE2, and 5′-AAC TTT GGC ATT GTG GAA GGG CTC-3′ and 5′-ACC CTG TTG CTG TAG CCG TAT TCA-3′ for GAPDH (internal control).
Statistical analysis was performed using two-tailed Student’s t-test or linear correlation analysis. P< 0.05 was considered significant.