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 plaq....

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 ....

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 associated .......

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

List of materials used in this article

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.