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.

The TgF344-AD rat line has been modified to model AD by expressing human APP and PS1_ΔE9 transgenes, leading to soluble and insoluble Aβ-40 and Aβ-42 expression and amyloid plaque formation⁵. It also presents the accumulation of hyperphosphorylated forms of the Tau protein leading to tauopathy. From the age of 9-24 months, the rats progressively develop the pathological hallmarks of AD and a cognitive impairment^5,6,7,8,9.

Positron Emission Tomography (PET), Single-Photon Emission computed Tomography (SPECT), and autoradiography are techniques based on the emission and quantification of γ rays. Radiotracers are quantified either in vivo (PET and SPECT) or ex vivo/in vitro (autoradiography). Those sensitive techniques have contributed to the understanding of mechanisms of several brain diseases, such as AD. Indeed, in terms of neuroinflammation, there are a lot of studies assessing 18 kDa Translocator Protein (TSPO), an in vivo neuroinflammation marker, with radiolabeled tracers such as [¹¹C]-(R)-PK11195 or [¹¹C]PBR28 (for review see¹⁰). In addition, alterations of neurotransmitter systems have been studied using radiotracers^11,12,13.

However, those techniques do not determine the cellular origin of the radioactive signal. This could hamper the interpretation of the biological underpinnings of the alteration in the binding of a radioligand in PET/SPECT. For instance, in the case of TSPO studies of neuroinflammation, understanding whether the increase or decrease of TSPO is due to astrocytic or microglial changes is of paramount importance. The Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT) technique was developed to get around these problems, allowing the assessment of radioligand binding in every cell type separately and the quantification of the target-protein density per cell. This innovative technique is consequently complementary and highly compatible with PET and SPECT imaging.

Here, this technique was applied along two axes: the study of neuroinflammation using TSPO-specific radioligands and assessing the serotonergic system. On the first axis, the aim was to understand the cellular origin of the TSPO signal in response to an acute inflammatory reaction. Therefore, FACS-RTT was used on the brain tissues of rats after the induction of neuroinflammation via a lipopolysaccharide (LPS) injection and following an in vivo [¹²⁵I]CLINDE SPECT imaging study. Further, the same imaging and FACS-RTT protocol were applied on 12- and 24-month-old TgF344-AD rats and matching wild-type (WT) rats. The second axis aimed to determine the origin of serotoninergic system alterations in this rat model via ex vivo 5-HT_2AR density assessment by cell type.

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 composed of one 10 min scan acquisition. Set the Step mode to Fine and the Acquisition mode to the Listmode (to record the entire emission spectrum).
3. Set up the bed and ensure that the warming system, breathing sensor, and anesthesia are functional and secure (Figure 1A-D). Then, place a phantom (i.e., 2 mL of a known concentration of ¹²⁵I in a 2 mL microfuge tube) where the animal's head will be positioned (centered on the area, Figure 1E).
4. Set the scan area by sliding the cursors for the three dimensions with the help of the three images in the bottom part of the screen. Ensure that the scan volume of the phantom and the animal is the same.
5. Start the phantom scan for subsequent calibration with the parameters established in steps 1.2-1.4.

2. Workspace setup for SPECT imaging

1. Clean the workspace with disinfectant virucide and place soft papers on all the surfaces.
2. Ensure that there is enough isoflurane and oxygen in their respective tanks.
3. Prepare a 24 G catheter by cutting off the fins of the butterfly catheter to have a clearer view of the rat tail vein.
4. Coat the catheter by filling it completely with heparin solution (25000 U/mL) after removing the needle. Then, place the needle back in it to avoid blood clot formation following catheter insertion.

3. [¹²⁵I]CLINDE radiotracer synthesis

CAUTION: Radioactivity can have sufficient ionizing energy to affect the atoms of the living cells and damage their genetic material (DNA).

1. Ensure to work in an appropriate environment, authorized for experiments involving radioactivity.
   NOTE: Wear the appropriate personal protective equipment (PPE), for radioactivity handling, including finger and body dosimeters. Try to stay at a safe distance from any source of radioactivity.
2. Incubate 100 µg of tributylin precursor in 100 µL of acetic acid with sodium iodide (Na¹²⁵I) (see Table of Materials) and 5 µL of 37% peracetic acid at 70 °C for 20 min in a thermocycler positioned in a glove box.
3. Dilute the reaction using 50% acetonitrile (ACN) in water to reach a volume of 500 µL. Inject the 500 µL of the diluted reaction onto a reverse-phase column (see Table of Materials).
4. Isolate the [¹²⁵I]CLINDE with a linear gradient HPLC run from 5%-95% ACN in 7 mM H₃PO₄ for 10 min. Dilute the isolate in H₂O to attain a final volume of 10 mL, and then inject the diluted reaction onto a concentration column (see Table of Materials).
5. Elute the [¹²⁵I]CLINDE from the column with 300 µL of absolute ethanol, and then evaporate the ethanol by incubating it in a vacuum centrifuge at RT for 40 min.
6. Dilute the residue containing the [¹²⁵I]CLINDE in 300 µL of sterile saline to create a stock solution.
7. After measuring its radioactivity, dilute the stock solution in sterile saline to obtain a solution of 0.037 MBq in 500 µL.
8. Purify the tracer by HPLC (High-performance liquid chromatography). Determine the elution time using a standard calibration at 450 nm, and isolate the single radioactive peak. Perform the standard calibration curve using seven standard concentrations of cold (non-radioactive) CLINDE (0.1 µg, 0.5 µg, 0.75 µg, 1 µg, 1.5 µg, 2 µg, and 5 µg in 400 µL of a solution of 50% ACN). Establish the radiochemical purity by measuring a single peak in radio TLC (Thin layer chromatography). Ensure that the purity of the radiochemical is above 60%.
9. Check the specific activity of the radioligand measuring ultraviolet absorbance at 450 nm and the calibration curves established with cold reference compounds. Ensure that the specific activity is greater than 1000 GBq/µmol.

4. [¹²⁵I]R91150 radiotracer synthesis

NOTE: Ensure to follow the same security rules as mentioned in the CLINDE synthesis section.

1. Mix 300 µg of R91150 precursor in a solution of 3 µL of absolute ethanol, 3 µL of glacial acetic acid, 15 µL of carrier-free Na¹²⁵I (see Table of Materials) (10 mCi) in 0.05 M NaOH and 3 µL of 30% H₂O₂. Incubate for 30 min in a glove box.
2. Inject the entire reaction into a reverse-phase column (see Table of Materials).
3. Isolate [¹²⁵I]R91150 by isocratic HPLC run (ACN/water 50/50, 10 mM acetic acid buffer) with a flow rate of 3 mL/min.
4. Dilute [¹²⁵I]R91150 in H₂O for a final volume of 10 mL.
5. Inject 10 mL of the diluted reaction onto a concentration column (see Table of Materials).
6. Elute [¹²⁵I]R91150 from the column with 300 µL of absolute ethanol.
7. Evaporate the ethanol by incubating it in a vacuum centrifuge at RT for 40 min.
8. Dilute the residue containing [¹²⁵I]R91150 in 300 µL of saline.
9. Purify the tracer by HPLC. Determine the elution time using a standard calibration at 450 nm and isolate the single radioactive peak. Establish the radiochemical purity by measuring a single peak in radio TLC. Ensure that the purity of the radiochemical is above 98%.

5. Animal preparation

1. Weigh and anesthetize the TgF344-AD rat (male or female, from 2-24 months old) in an induction chamber with 3% isoflurane. Once deeply anesthetized, lower the isoflurane flow to 2% (0.4 L/min, 100% O2) in the chamber.
2. Position the animal on a pre-warmed bed equipped with an anesthesia nosecone. Maintain isoflurane at 2% (0.4 L/min, 100% O₂).
3. Apply eye gel lubricant in the animal's eyes and confirm the depth of anesthesia via respiratory monitoring; adjust anesthesia if necessary.
4. Perform the 24 G catheter insertion in the tail vein.

6. SPECT acquisition

1. Transfer the animal to the camera bed equipped with a temperature-controlled heating pad set to 38°C (Figure 1E).
2. Secure the head of the animal onto the bite bar and fix the head supports.
3. Set up the experiment on a 60 min scan constituting 60 frames of 1 min. Reuse all other parameters set up in step 1. Click on the Update Image button to update the animal position.
4. Set the scan area by sliding the cursors with the help of the three images in the bottom part of the screen for the three dimensions.
5. Inject 500 µL of the radioactive radiotracer ([¹²⁵I]CLINDE or [¹²⁵I]R91150), and then flush the tube with 300 µL of sterile 0.9% NaCl. Simultaneously click on Start Acquisition to start the scan.
6. During the scan time, ensure to maintain the animal under constant anesthesia with respiration rate monitoring. Adjust the flow of isoflurane if needed.
7. Once the scan is over, quickly euthanize the anesthetized animal by decapitation.

7. Scan reconstruction

1. Open the scan reconstruction software (see Table of Materials), and then open the data set, looking for the [filename].parameters file, created in the folder of the scan.
2. Select the isotope of interest. Note that the Listmode parameter allows multiple isotope selection at this step.
3. Select the following parameters: 0.4 mm Voxel size, 4 (POS-EM) subsets, 6 iterations (24ME-EM equivalent), no post-filter, and the isotope corresponding Decay Correction. Select the output format to NIfTI, and then select Start SPECT reconstruction.

8. Rat brain extraction

1. In the same anesthetic event as the scanning procedure and after ensuring the deep anesthesia state of the animal by monitoring its respiratory rate, proceed to the decapitation by guillotine and quickly transfer the head to the dissection bench.
2. With scissors, carefully cut the skin on top of the head from the back to the front till the middle of the eyes.
3. Cut off the excess muscles around the base of the skull and the cervical vertebras.
4. Next, carefully place one blade of the scissors in the hole at the back of the skull, the foramen magnum, and remove the back part of the skull with surgical pliers.
5. Then, with surgical pliers, carefully remove the top part of the skull. The skull of old male rats can be thick, remove as small pieces to avoid damage to the brain.
6. Carefully cut the meninges with scissors. Meninges can damage the brain during the extraction process; remove it as a precaution.
7. After removing the top part of the skull, turn the head of the animal around, and with a small flat spatula, carefully pull the brain out by cutting the optic and trigeminal nerves.
8. Carefully transfer the brain onto a flat, clean glass surface for dissection on ice.
9. Use a flat metal spatula and a razor blade to dissect the regions of interest of the brain. Place the tissues in a 2 mL centrifuge tube and weigh the tissue obtained. Use the brain section directly for cell isolation or quickly freeze it in liquid nitrogen for later use.

9. Cell isolation

1. Make sure to work in a clean and sterile environment. Working under a class-II biosafety cabinet (BSC) is advised. Ensure that the gloves and every piece of equipment introduced into the BSC are sterile.
2. To prepare the cells for cell sorting, follow the protocol from Jaclyn M. Schwarz¹⁴.
   NOTE: In this experiment, a commercially available Neural dissociation kit (see Table of Materials) was used for cell preparation.
   1. Put the samples into a 2 mL centrifuge tube with 1 mL of HBSS (Ca- and Mg-free), and then centrifuge (300 x g, 2 min, room temperature = RT) and remove the supernatant without disturbing the pellet.
   2. Add 1900 µL of enzyme mix-1 and incubate it for 15 min at 37 °C while agitating the tubes by inversion every 5 min.
   3. Add 30 µL of enzyme mix-2, agitate gently with pipette 1 (see Table of Materials) back and forth 30 times. Then, incubate for 15 min at 37 °C while agitating the tubes by inversion every 5 min.
   4. Mix gently back and forth with pipette 2, and then pipette 3 before incubating for 10 min at 37 °C to dissociate the tissues.
   5. Filter the cells with an 80 µm cell strainer, add 10 mL of HBSS (Ca- and Mg-free). Centrifuge (300 x g, 10 min, RT) and remove the supernatant without disturbing the pellet.
   6. For myelin depletion, resuspend the pellet with 400 µL of myelin removal buffer (see Table of Materials), and then add 100 µL of myelin removal beads (see Table of Materials), before incubating for 15 min at 4 °C.
   7. Add 5 mL of myelin removal buffer, centrifuge (300 x g, 10 min, RT), and remove the supernatant without disturbing the pellet.
   8. Add 500 µL of myelin removal buffer and place the tube into the magnetic field column. Wash the column with 1 mL of myelin removal buffer four times.
   9. Centrifuge (300 x g, 2 min, RT) and remove the supernatant without disturbing the pellet. Vortex briefly (2 s) to dissociate the cells and add 5 µL of Fc block CD32. Vortex again and incubate for 5 min at 4 °C.
   10. Add 100 µL of the mix of primary antibodies of interest; incubate for 20 min at 4 °C.
   11. Centrifuge (350 x g, 5 min, 4 °C) and remove the supernatant without disturbing the pellet. Blot the tubes upside-down on soft paper.
   12. After a brief vortex (2 s), add 100 µL of the secondary antibody mix and incubate for 15 min at 4 °C.
   13. Add 2 mL of myelin removal buffer, centrifuge (350 x g, 5 min, 4 °C) and remove the supernatant without disturbing the pellet. Blot the tubes upside-down on soft paper. Resuspend the cells in 250 µL of sterile PBS and proceed directly to cell sorting.

10. Cell sorting

1. Prepare the microfuge tube with 500 µL of sterile PBS for collecting the sorted cells.
2. Add 10 µL of Hoechst per 1000 µL of cell solution to color the nuclei of the living cells and distinguish them from the dead cells.
3. Transfer the cells as soon as possible to the cell sorting machine at 4 °C.
4. First, sort the cells by forward and side scatter, and then sort the Hoechst positive cells. Next, sort the cells based on the antibodies of interest. Collect the positively stained cells separately. Make sure to distinguish the positive cells from the autofluorescent ones.
5. Count the number of sorted cells for each pool of interest.

11. Gamma counting

1. Calibrate the γ counting system with 10 µL of the same phantom solution used for the SPECT calibration but dilute it in 1 mL of water.
   NOTE: The phantom solution is diluted because of the enhanced precision of the γ counting system.
2. Place the tube of sorted cells in the γ counting system and proceed with the γ counting according to the manufacturer's protocol.

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

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