Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Summary

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-β (Aβ) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer's disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Abstract

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-β (Aβ) phagocytosis by mononuclear phagocytes in rodent Alzheimer's Disease (AD) models. The method involves fluorescently visualizing Aβ encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of Aβ spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Introduction

Alzheimer's Disease (AD), the most common age-related dementia¹, is characterized by cerebral amyloid-β (Aβ) accumulation as "senile" β-amyloid plaques, chronic low-level neuroinflammation, tauopathy, neuronal loss, and cognitive disturbance². In AD patient brains, neuroinflammation is earmarked by reactive astrocytes and mononuclear phagocytes (referred to as microglia, although their central vs. peripheral origin remains unclear) surrounding Aβ deposits³. As the innate immune sentinels of the CNS, microglia are centrally positioned to clear brain Aβ. However, microglial recruitment to Aβ plaques is accompanied by very little, if any, Aβ phagocytosis^4,5. One hypothesis is that microglia are initially neuroprotective by phagocytozing small assemblies of Aβ. However, eventually these cells become neurotoxic as overwhelming Aβ burden and/or age-related functional decline, provokes microglia into a dysfunctional proinflammatory phenotype, contributing to neurotoxicity and cognitive decline⁶.

Recent Genome-wide Association Studies (GWAS) have identified a cluster of AD risk alleles belonging to core innate immune pathways⁷ that modulate phagocytosis^8-11. Consequently, the immune response to cerebral amyloid deposition has become a major area of interest, both in terms of understanding AD etiology and for developing new therapeutic approaches^12-14. Yet, there is a vital need for methodology to evaluate Aβ phagocytosis in vivo. To address this unmet need, we have developed quantitative 3D in silico modeling (q3DISM) to enable true 3D quantitation of cerebral Aβ phagocytosis by mononuclear phagocytes in rodent models of Alzheimer-like disease.

Limited only by the extent to which they recapitulate disease, animal models have proven invaluable for understanding AD pathoetiology and for evaluating experimental therapeutics. Owing to the fact that mutations in the Presenilin (PS) and Amyloid Precursor Protein (APP) genes independently cause autosomal dominant AD, these mutant transgenes have been extensively used to generate transgenic rodent models. Transgenic APP/PS1 mice simultaneously coexpressing "Swedish" mutant human APP (APP_swe) and Δ exon 9 mutant human presenilin 1 (PS1ΔE9) present with accelerated cerebral amyloidosis and neuroinflammation^15,16. Further, we have generated bi-transgenic rats coinjected with APP_swe and PS1ΔE9 constructs (line TgF344-AD, on a Fischer 344 background). Unlike transgenic mouse models of cerebral amyloidosis, TgF344-AD rats develop cerebral amyloid that precedes tauopathy, apoptotic loss of neurons, and behavioral impairment¹⁷.

In this report, we describe a protocol for immunostaining microglia, phagolysosomes and Aβ deposits in brain sections from APP/PS1 mice and TgF344-AD rats, and acquisition of large z-dimensional confocal images. We detail in silico generation and analysis of true 3D reconstructions from confocal datasets allowing quantitation of Aβ uptake into microglial phagolysosomes. More broadly, the methodology that we detail here can be used to quantify virtually any form of phagocytosis in vivo.

Protocol

Statement of research ethics: All experiments involving animals detailed herein were approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC) and performed in strict accordance with National Institutes of Health guidelines and recommendations from the Association for Assessment and Accreditation of Laboratory Animal Care International.

1. Rodent Brain Isolation and Preparation for Immunostaining

DAY 1:

1. Place aged TgF344-AD rats (14-month-old) or APP/PS1 mice (12-month-old) under continuous deep isoflurane anesthesia (4%). Assess the depth of anesthesia by toe pinch and the absence of withdrawal reflex.
2. Cut through both sides of the rib cage and lift to expose the heart. Insert a 23 G needle into the left ventricle of the heart and make a small incision into the right atrium. Proceed to exsanguination by transcardial perfusion with ice-cold phosphate-buffered saline (PBS) using a peristaltic pump (30 mL for mice; 150 - 200 mL for rats).
3. Make a caudal midline incision into the skin and move the skin and muscle aside. Cut through the top of the skull along the midline and between the eyes. Remove the bone plates and isolate the whole brain from the skull.
4. Place the brain into a coronal rodent brain matrix and slice it into quarters. Incubate posterior quarters O/N (16 h) in paraformaldehyde fixative (4% PFA in PBS) at 4 °C. Wash 3x in PBS, and then transfer to 70% ethanol. Caution: PFA is toxic and should be handled under a chemical hood with appropriate personal protection equipment.

DAY 2:

5. Place brain quarters into embedding cassettes and progressively dehydrate tissue in successively more concentrated 1 h ethanol baths (70%, 80%, 95%, and 100% x3).
6. Clear ethanol from the tissue with three successive 100% xylene baths (1 h each). Caution: Xylene is toxic and should be handled under a chemical hood with appropriate personal protection equipment.
7. Embed tissue in paraffin blocks after two molten paraffin wax baths (56 - 58 °C, 90 min each).

DAY 3:

8. Cut 10 μm-thick sections of paraffin-embedded brains using a microtome. Dip sections into a water bath (50 °C for 1 min) and apply to microscope slides. Leave the slides to dry O/N, ensuring tissue adhesion to the slide.

2. Immunostaining

Note: Different combinations of antibodies can be utilized for the staining procedure described below. Antibody cocktails compatible with brain tissue from rats and mice are listed in Table 1.

DAY 4:

1. Deparaffinize brain sections using 2x 100% xylene baths (12 min each).
2. Rehydrate brain sections in successive ethanol baths — 100% for 10 min, 95% for 5 min, 80% for 10 min, and finally 70% for 15 min — followed by 3x PBS washes [5 min at RT, with light agitation]. Meanwhile, heat antigen retrieval solution to 95 - 97 °C on a hot plate with magnetic bar stirring.
3. Incubate brain sections in antigen retrieval solution at 95 - 97 °C for 30 min. Then, wash 3x in PBS (5 min at RT, with light agitation).
4. Quickly dry slides using delicate task wipers to avoid tissue drying, and draw a hydrophobic barrier around the tissue area with a hydrophobic barrier pen. Fill the encircled tissue region with blocking buffer [PBS containing 0.3% Triton X-100 and 10% Normal Donkey Serum (NDS)], and incubate at RT for 1 h in a humidified chamber.
5. Replace blocking buffer with Iba1 primary antibody (diluted in blocking buffer) to label mononuclear phagocytes, and incubate O/N at 4 °C in a humidified chamber. For antibody hosts and working dilutions, see Table 1 and the Table of Materials.

DAY 5:

6. Rinse primary antibody with 3x PBS baths (5 min at RT with light agitation). Incubate with fluorescent secondary antibody (conjugated with a 594 nm emission fluorophore) for 1 h (in blocking buffer at RT in the dark) followed by 3x PBS baths (5 min at RT with light agitation). At this time, maintain sections in the dark to avoid fluorescent signal bleaching.

DAY 6 - 7:

7. Repeat steps 2.5 & 2.6 with CD68 (rat brains) or LAMP1 (mouse tissue) antibodies and appropriate secondary antibodies (coupled with a 488 nm emission fluorophore) to label phagolysosomes.

DAY 7 - 8:

8. Repeat steps 2.5 & 2.6 with OC (rat tissue) or 4G8 (mouse brains) antibodies and appropriate secondary antibodies (coupled to a 647 nm fluorophore) to label Aβ deposits.
   NOTE: Alternatively, 6E10 antibody can be used successfully both on mouse and rat tissue. For appropriate antibody combinations, see the Table of Materials.
9. Allow sections to completely dry O/N at RT in the dark. Then, cover specimens with a cover slip sealed by fluorescence mounting media containing DAPI.

3. Acquisition of Large Z-stack Confocal Datasets

Note: This protocol requires a fully automated laser scanning confocal microscope equipped with a 60X objective and 405 nm, 488 nm, 594 nm, and 647 nm lasers. All equipment is computer controlled by imaging and laser control software. Prior to beginning the imaging protocol, power on the computer, epifluorescent lamp, microscope, lasers and camera.

DAY 9:

1. Select the 60X microscope objective. Add immersion oil to the lens, and place the sample onto the microscope stage slide holder. Raise the objective until the oil makes contact with the slide. Adjust the focal plane to locate amyloid plaques in the hippocampus or cerebral cortex using epifluorescent illumination through the oculars.
2. Acquire confocal images of activated mononuclear phagocytes surrounding amyloid deposits in the hippocampus or cortex of rodents by confocal microscopy (60X magnification, z-stack steps: 0.25 μm < z < 0.40 μm, number of steps 25 < n < 35).

4. q3DISM

Note: In order to yield significant results, we suggest analyzing a minimum of 3 images per animal/region of interest. For each image, the abundance of cells to analyze may vary depending on experimental paradigms. In the representative results shown in this report, we analyzed 3 cells/condition (e.g., mononuclear phagocytes distant from or associated with plaques; see Figures 1C - D and 2C - D).

DAY 10:

1. Analyze confocal datasets with scientific 3D image processing and analysis software colocalization (coloc) module for spatial proximity of Iba1/CD68 (rat tissue) or Iba1/LAMP1 (mouse tissue) staining in all z-planes simultaneously. Create Iba⁺/CD68⁺ or Iba⁺/LAMP1⁺ colocalization channels that correspond to phagolysosomes within activated mononuclear phagocytes.
   1. Select TRITC for channel A (corresponding to Iba1 staining coupled with 594 nm fluorophore) and FITC for channel B (corresponding to CD68 or LAMP1 staining coupled with 488 nm fluorophore). On the right-hand side of the software window for 'mode check,' select 'threshold,' and for 'coloc intensities,' select 'source channels'.
   2. Click 'Edit' to select 'coloc color' on the right-hand side of the software window.
   3. For each channel independently, adjust thresholds to include specific staining and exclude background/non-specific signals. Once adjusted, do not change thresholds between images to ensure an unbiased analysis. The colocalized voxels (pixels from all z-stacks) will appear in the color selected in step 4.1.2 in all z-stacks simultaneously.
   4. Click 'build coloc channel'. The colocalization channel created will appear in the display adjustment window.
   5. Click on the coloc channel to open channel statistics. The '% of volume/material A above threshold colocalized' represents the % of Iba1 signal (voxels corresponding to the 594 nm fluorophore) colocalized with LAMP1 or CD68 signal (voxels corresponding to the 488 nm fluorophore). More simply, this is the monocyte volume occupied by phagolysosomes (see Figures 1C and 2C).
      NOTE: '% of volume/material B above threshold colocalized' corresponds to % of LAMP1 or CD68 signal colocalized with Iba1. This should be close to 100%, as phagolysosomes are intracellular structures. Values are based on ratio of signal from all z-stacks above threshold colocalized.
2. Using the coloc module, analyze the coloc channel created in step 4.1 for spatial proximity with OC (rat tissue) or 4G8 (mouse tissue) Aβ signals. This allows for quantitation of Aβ encapsulated within phagolysosomes.
   1. Select the channel A coloc dataset (corresponding to the Iba1/CD68 or Iba1/LAMP1 coloc channel created in step 4.1) and Cy5 for channel B (corresponding to OC or 4G8 staining coupled with 647 nm fluorophore).
   2. Build a coloc channel as described in steps 4.1.2 to 4.1.5.
   3. Click on the coloc channel to open channel statistics. The '% of volume/material A above threshold colocalized' represents the % of Iba1/LAMP1 or Iba1/CD68 (voxels corresponding to the coloc channel built in step 4.1.5.) colocalized with OC or 4G8 signal (voxels corresponding to the 647 nm fluorophore). This is the phagolysosomal volume occupied by Aβ (see Figures 1D and 2D).
      NOTE: '% of volume/material B above threshold colocalized' corresponds with % of total Aβ signal colocalized with phagolysosomes. This can be used to evaluate the fraction of total Aβ deposits encapsulated within phagolysosomes (not shown in the present representative results).
3. Use the surpass module to reconstruct the confocal image stacks and generate 3D models of Aβ encapsulated within monocyte phagolysosomes.
   1. In the 'display adjustment' window, select TRITC (to show only Iba1 staining). In the 'volume properties' window, click 'add new surface'.
   2. In step '1/5 algorithm', in 'settings,' select 'surface.' Also, in 'color,' select color type in palette or RGB and adjust transparency (red is set at 60% transparency in Figures 1B and 2B). Check box 'select region of interest'. Click next.
   3. In Step '2/5 region of interest,' draw a window around the cell of interest by adjusting x, y and z coordinates (see white boxes in Figures 1A and 2A). Click next.
   4. In Step '3/5 source channel', select the TRITC source channel. Check the box 'smooth,' and set surface area detail level to 0.4 μm. For 'thresholding,' select absolute intensity. Click next.
   5. In Step '4/5 threshold,' adjust threshold so that the volume created overlaps perfectly with the TRITC channel signal. Click next.
   6. In Step '5/5 classify surfaces,' in section 'filter type,' select objects depending on their size to be included or excluded from the volumes to be created. Click finish, execute all creation steps, and exit the wizard.
   7. Repeat steps 4.3.1 to 4.3.6 to create a 3D surface for the FITC channel (LAMP1⁺ or CD68⁺ phagolysosomes).
   8. Repeat steps 4.3.1 to 4.3.6 to create a 3D surface for the Cy5 channel (OC⁺ or 4G8⁺ Aβ deposits).

Results

Using the multi-stage methodology for q3DISM detailed above, we are able to quantify Aβ uptake into monocyte phagolysosomes in the brains of APP/PS1 mice (Figure 1) and TgF344-AD rats (Figure 2). Therefore, the q3DISM methodology has enabled analysis of mononuclear phagocytes in mouse and rat models of AD. Interestingly, the volume occupied by CD68⁺ phagolysosomes is significantly increased in Iba1⁺ mononuclear phagocytes associ...

Discussion

The protocol that we describe in this report for true 3D quantitation of Aβ phagocytosis in vivo by mononuclear phagocytes relies on specific labeling of cellular and subcellular compartments as well as Aβ deposits. Specifically, we use Iba1 (Ionized-calcium Binding Adaptor molecule 1), a protein that is involved in membrane ruffling and phagocytosis upon cell activation^18,19, to stain cerebral mononuclear phagocytes. While Iba1⁺ cells are generally regarded ...

Materials

Name	Company	Catalog Number	Comments
Isoflurane	Abbott	NDC 0044-5260-05
Dissecting scissors	VWR	82027-582
Dissecting scissors Blunt tip	VWR	82027-588
Tweezers	VWR	94024-408
23 G needle	VWR	BD305145
peristaltic pump FH10	Thermo Scientific	72-310-010
PBS 10x	Bioland Scientific	PBS01-02	Phosphate-buffered Saline; Working concentration 1x
Adult Mouse Brain Matrix, Coronal slices, Stainless Steel 1 mm	Kent Scientific	RBMS-200C
Adult Rat Brain Matrix, Coronal slices, Stainless Steel 1 mm	Kent Scientific	RBMS-305C
32% Paraformaldehyde aqueous solution (PFA)	EMS	15714-S	Caution: Toxic. Working concentration: 4% in PBS
Ethanol	VWR	89125-188	Various concentrations, see protocol
Tissue-Tek Uni-cassettes Sakura	VWR	25608-774
Embedding and Infiltration Paraffin	VWR	15147-839
Microtome Leica RM2125	Leica Biosystems
Disposable Microtome Blades	VWR	25608-964
Water bath Leica HI 1210	Leica Biosystems
Micro slide Superfrost plus	VWR	48311-703
Xylene	Sigma-Aldrich	534056-4X4L	Caution: Toxic
Target Retrieval Solution 10x	DAKO	S1699	Working concentration 1x
KimWipes	VWR	21905-026
Hydrophobic PAP pen	VWR	95025-252
Triton X-100	VWR	97062-208
Normal Donkey Serum (NDS)	Jackson Immuno	017-000-121
Coverslips	VWR	48393081
Prolong Gold antifade reagent with DAPI	Life Technologies	P36935
Glass Slide Rack	VWR	100492-942
Iba1 antibody (polyclonal, rabbit)	Wako	019-19741	Working concentration 1:200
Iba1 antibody (polyclonal, goat)	LifeSpan Bioscience	LS-B2645	Working concentration 1:200
rat CD68 [KP1] antibody (monoclonal, mouse)	Abcam	ab955	Working concentration 1:200
mouse CD68 [FA-11] antibody (monoclonal, rat)	Abcam	ab53444	Working concentration 1:200
mouse CD107a (LAMP1) antibody (monoclonal, rat)	Affymetrix	14-1071	Working concentration 1:100
Beta-Amyloid, 17 - 24 (4G8) antibody (monoclonal, mouse)	Covance	SIG-39220	Working concentration 1:200
Beta-Amyloid, 1 - 16 (6E10) antibody (monoclonal, mouse)	Covance	SIG-39320	Working concentration 1:200
OC antibody (polyclonal, rabbit)	Gifted by D. H. Cribbs and C. G. Glabe (UC Irvine)	Working concentration 1:200
Alexa Fluor 488 mouse secondary antibody	Invitrogen	A-11001	Working concentration 1:1,000
Alexa Fluor 488 rat secondary antibody	Invitrogen	A-11006	Working concentration 1:1,000
Alexa Fluor 594 rabbit secondary antibody	Invitrogen	A-11037	Working concentration 1:1,000
Alexa Fluor 594 goat secondary antibody	Invitrogen	A-11080	Working concentration 1:1,000
Alexa Fluor 647 mouse secondary antibody	Invitrogen	A-21235	Working concentration 1:1,000
Alexa Fluor 647 rabbit secondary antibody	Invitrogen	A-21443	Working concentration 1:1,000
Immersion oil	Nikon
A1 Confocal microscope	Nikon
NIS Elements Advanced Research software	Nikon
Imaris:Bitplane software version 7.6	Bitplane		"coloc" and "supass" modules are used. Alternatively, the open-source freeware ImageJ can be used for colocalization analysis of confocal z-stacks datasets.