Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

Summary

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) is a powerful tool to study the role of the 18 kDa translocator protein or Serotonin 5HT_2A-receptor expression in Alzheimer's Disease at a cellular scale. This protocol describes the ex-vivo application of FACS-RTT in the TgF344-AD rat model.

Abstract

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their alterations are perhaps associated with a pro-inflammatory state. The TgF344-AD rat strain has been designed to express human APP and human PS1_ΔE9 genes, encoding for amyloid proteins Aβ-40 and Aβ-42 and displays amyloid pathology and cognitive deficits with aging. The TgF344-AD rat model is used in this study to evaluate the cellular origin of the 18 kDa translocator protein (TSPO, a marker of glial cell activation) binding, and the 5HT_2A-receptor (5HT_2AR) serotonin receptor levels that are possibly disrupted in AD. The technique presented here is Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT), a quantitative cell-type-specific technique complementary to in vivo PET or SPECT or ex vivo/in vitro autoradiography techniques. It quantifies the same radiolabeled tracer used prior for imaging, using a γ counter after cytometry cell sorting. This allows determining the cellular origin of the radiolabeled protein with high cellular specificity and sensitivity. For example, studies with FACS-RTT showed that (i) the increase in TSPO binding was associated with microglia in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation, (ii) an increase in TSPO binding at 12- and 18-months was associated with astrocytes first, and then microglia in the TgF344-AD rats compared to wild type (WT) rats, and (iii) the striatal density of 5HT_2AR decreases in astrocytes at 18 months in the same rat AD model. Interestingly, this technique can be extended to virtually all radiotracers.

Introduction

Neurodegenerative diseases, such as Alzheimer's Disease (AD), are characterized by a neuronal loss associated with increased symptoms. AD, the most common cause of dementia, accounting 60%-70% of cases, affects around 50 million people worldwide¹. At a neuropathological level, the two major characteristics of AD are the accumulation of extracellular amyloid-β (Aβ) plaques and intracellular Tau neurofibrillary tangles. Glial cell alterations have also been associated with AD² and possible disruption of several neurotransmitter systems^3,4.

Protocol

All experimental procedures were conducted in agreement with the Ethics Committee for Human and Animal Experimentation of the Canton of Geneva, the Cantonal Commission for Research Ethics (CCER), and the General Direction Of The Health Of The Canton Of Geneva (Switzerland), respectively. Data are reported following Animal Research: Reporting In-vivo Experiments (ARRIVE) guidelines.

1. SPECT camera preparation and calibration

1. Turn on the camera, load the operating software (see Table of Materials). Click on the Home XYZ Stage button to perform homing.
2. Set up the experiment comp....

Results

WT rats experienced in vivo SPECT scan with [¹²⁵I]CLINDE radiotracer after a unilateral LPS injection (Figure 2). This scan (using summed data from images of 45-60 min post radiotracer injection) showed higher binding of [¹²⁵I]CLINDE in the site of the LPS injection (Figure 2A) than in the contralateral region of the brain (Figure 2B). The ex vivo samples that underwent FACS-RTT confirmed thos.......

Discussion

To our knowledge, this technique was the first to describe an approach that allows a better understanding of in vivo binding alterations of a radiotracer at the cellular level. The protocol describes a multiscale method to quantify radiotracer binding at the cellular level using [¹²⁵I]CLINDE (TSPO) or [¹²⁵I]R91150 (5HT_2AR) as examples.

This technique is robust and sensitive enough to precisely detect the cellular origin of a wide spectrum of glial cell.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Sigma-Aldrich
Acetonitrile	Sigma-Aldrich
BioVet	BioVet		Software for vitals check
Bondclone C18 reverse-phase column	Phenomenex, Schlieren, Switzerland
Des-Sur	University Hospital of Geneva		Virucide
Fc Block / anti-CD32	BD Biosciences	BDB550270	Reactivity for rat
FITC-conjugated anti-rat CD90	Biolegend	202504	Reactivity for rat
Heparin	B. Braun	B01AB01
HPLC	Knauer
Insyte-W 24 GA 0.75 IN 0.7 x 19 mm	BD Biosciences	321312	24 G catheter
Isoflurane	Baxter	ZDG9623
Lacryvisc	Alcon	2160699
LS Columns	Miltenyi Biotec	130-042-401
MACS MultiStand	Miltenyi Biotec	130-042-303
Micropore soft tape	3M	F51DA01
MILabs-Uspect II	MILabs		Software for SPECT Camera
MoFlo Astrios	Beckman Coulter		Cell sorter
Myelin Removal Beads II	Miltenyi Biotec	130-096-733	Contains beads and myelin removal buffer.
NaCl 0.9% Sterile solution	B. Braun	395202
Neural Dissociation Kit (P)	Miltenyi Biotec	130-092-628	Contains the enzyme mixes, pipets 1, 2 and 3.
Nylon Mesh Sheet	Amazon	CMN-0074-10YD	40 inch width, 80 micron size mesh
Peracetic acid	Sigma-Aldrich
QuadroMACS Separator	Miltenyi Biotec	130-090-976
R91150 précursor	CERMN
Sep-Pak C18 Column	Waters		Concentration column
Sodium iodide Na125	PerkinElmer
Tributylin precursor	CERMN
U-SPECT Rec2.38c	MILabs	Version Rec2.38c	Software for SPECT images reconstruction
USPECT II	MILabs		Spect Camera
Wizard 3"	PerkinElmer		Gamma counter