Fluorine (F) MRI for Assessing Inflammatory Cells in the Kidney: Experimental Protocol

Inflammation is one underlying contributing factor in the pathology of acute and chronic kidney disorders. Phagocytes such as monocytes, neutrophils and dendritic cells are considered to play a deleterious role in the progression of kidney disease but may also contribute to organ homeostasis. The kidney is a target of life-threatening autoimmune disorders such as the antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV). Neutrophils and monocytes express ANCA antigens and play an important role in the pathogenesis of AAV. Noninvasive in vivo methods that can quantify the distribution of inflammatory cells in the kidney as well as other organs in vivo would be vital to identify the causality and significance of inflammation during disease progression. Here we describe an noninvasive technique to study renal inflammation in rodents in vivo using fluorine (F) MRI. In this protocol we chose a murine ANCAAAV model of renal inflammation and made use of nanoparticles prepared from perfluoro-5-crown-15ether (PFCE) for renal F MRI. This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This experimental protocol chapter is complemented by two separate chapters describing the basic concept and data analysis.


Introduction
Inflammation is one underlying contributing factor in the pathology of acute and chronic kidney disorders [1]. Studies also suggest that systemic inflammation can cause ischemic injury in a vital organ, such as the kidney, which could then result in repercussions in another distant organ downstream of the ischemic event, such as the heart [2][3][4]. Early inflammatory events governed by cells of the innate immune system, such as macrophages, probably promote renal tissue injury but may also support repair [5][6][7][8]. Phagocytes such as dendritic cells (DC) and macrophages are considered to play a deleterious role in the inflammatory outcome of chronic kidney disease but may also contribute to organ homeostasis [9].
The kidney is often a target of systemic autoimmune disorders that are compounded by complex inflammatory processes [10]. Examples of life-threatening autoimmune disorders that affect the kidneys are the antineutrophil cytoplasmic antibody (ANCA)associated vasculitides (AAV) manifesting as rapidly progressive necrotizing crescentic glomerulonephritis (NCGN) [11]. Neutrophils and monocytes express ANCA antigens and ANCA induces neutrophil extracellular traps that cause RIPK1-dependent endothelial cell (EC) damage via activation of the alternative complement pathway [12]. While the central role of neutrophil activation in ANCAassociated vasculitis and NCGN is clear, the role of monocytes/ macrophages was only recently uncovered in a renal ANCAassociated vasculitis model [13].
Noninvasive in vivo methods that can quantify the level of inflammation in the kidney as well as other organs in system autoimmune disorders such as ANCA-associated vasculitides would be vital to identify the causality and significance of inflammation during the course of disease. One method to visualize inflammation by MRI makes use of MR contrast agents that modulate T 2 * and that are easily taken up by phagocytic inflammatory cells [14,15]. Iron oxide particles including ultrasmall iron oxide agents (USPIO) have been used as susceptibility (T 2 * ) MR contrast agents to target inflammatory cell populations. These particles are engulfed by phagocytic cells in the blood. Drawbacks of USPIO-based T 2 * studies include MR signal quantification and difficulty to distinguish contrast created by labeled cells from other intrinsic tissue contrasts [14].
Here we describe an alternative noninvasive technique to study inflammation in rodents in vivo using fluorine ( 19 F) MRI. 19 F MRI is performed in association with intravenous injections of perfluorocarbon (PFC) nanoparticles (NPs). These NPs are taken up by cells of the immune system traveling through the circulation into the inflammatory regions. Thus 19 F MRI is ideal for studying distribution of inflammatory cell in vivo. In this protocol we chose a murine AAV model of renal inflammation that has been described in greater depth elsewhere [16] and made use of nanoparticles prepared from perfluoro-5-crown-15-ether (PFCE).
This experimental protocol chapter is complemented by two separate chapters describing the basic concept (please see the chapter by Waiczies S et al. "Functional Imaging Using Fluorine ( 19 F) MR Methods: Basic Concepts") and data analysis (please see the chapter by Starke L et al. "Data Preparation Protocol for Low Signal-to-Noise Ratio Fluorine-19 MRI"), which are both part of this book.
This chapter is part of the book Pohlmann A, Niendorf T (eds) (2020) Preclinical MRI of the Kidney-Methods and Protocols. Springer, New York.

Animals
This experimental protocol is tailored for mice with a body mass of 20-30 g (e.g., wild type C57BL/6 mice or a disease model of renal inflammation). Here we describe briefly how to generate the AAV animal model. More thorough detail on the immunization, bone marrow transplantation as well purification of mouse MPO is given in the study establishing the MPO-AAV animal model [16]. Wild-type (WT) C57BL/6J mice (B6) (Jackson Laboratories, Bar Harbor, ME) and myeloperoxidase-deficient (MPO À/À ) mice (generated by Aratani et al. [17]) were used in this protocol. MPO À/À mice were immunized with murine MPO at the age of 8-10 weeks, subjected to lethal irradiation, and then transplanted with MPO-expressing bone marrow cells. Animal experiments should be approved by animal welfare authorities and guidelines to minimize discomfort to animals (86/609/EEC). 5. Mouse sled with a breathing mask connected to the isoflurane system.

MRI Techniques
Typically, 19 F MR studies applying PFC compounds such as PFCE to study inflammation in vivo employ the turbo spin echo (SE) rapid acquisition using relaxation enhancement (RARE) sequence [18][19][20][21][22]. This method reduces acquisition time by accumulating multiple echoes within a single repetition time [23]. Typically T 1 of these 19 F compounds is in the range of 0.5-3 s, depending on the compound and also magnetic field strength (B 0 ). However, if employing paramagnetic macrocyclic PFC compounds complexed to lanthanides, T 1 values can be reduced to the order of 1-15 ms and T 2 * values correspondingly reduced to 0.4-12 ms and a radial zero echo time (ZTE) sequence might be better suited [24].
2. 19 F spectroscopy sequence: Block pulse for nonlocalized (global) is a standard sequence on Bruker MRI systems, where it is called "SINGLEPULSE" (see Note 3).

19 F MR Imaging
Typically T 1 of PFC compounds is in the range of 0.5-3 s, depending on the compound and also magnetic field strength (B 0 ). When working with a standard diamagnetic PFC such as PFCE that is documented in the literature, it is recommended that the T 1 and T 2 of the compound be studied at 37 C before starting with the first in vivo experiments to study inflammation. According to the measured relaxation times (T 1 /T 2 ), the optimal settings for RARE, namely echo train lengths (ETL) and repetition time (TR) should be calculated to improve sensitivity thresholds. 2. NP characterization: Measure mean particle size (in nm), polydispersity index (PdI), and zetapotential (mV) by using a dynamic light scattering machine such as the one listed above. Use the z-average diameter for particle size since it gives an intensity-weighted harmonic diameter and is ideal for comparing different analyses. The nanoparticles should have a PdI < 0.3 indicating a relatively low polydispersity and narrow size distribution (see Note 8).
3. NP application: Administer 19 F nanoparticles via tail vein at a dose of 5-80 μmol of PFCE molecules, depending on the frequency of the bolus injections. Start administering 19 F nanoparticles at relevant time-points of your inflammatory model, for example, in MPO immunized MPO À/À mice subjected to lethal irradiation we started intravenous application of PFCE nanoparticles 4-8 weeks following transplantation of MPO-expressing bone marrow cells (see Note 9).

Preparation Prior to 19 F/ 1 H MRI Scans
Four to 18 h following the last intravenous administration of 19 F nanoparticles prepare the mice for 19 F/ 1 H MRI: 1. First anesthetize the mice by inhalation narcosis using a mouse chamber connected to a isoflurane inhalation system and gas-mixing system (see Note 10).
2. Adjust the flow rate for air and O 2 at 0.2 and 0.1 l/min respectively and 3% isoflurane (adjusted from a vaporizer) for about 2 min until the required level of anesthesia is reached (no response following toe pinch).
3. Transfer mice to the MR scanner. Should a quantification of inflammation be required, a reference tube with a known concentration of 19  3. Deselect the automatic reference gain (RG) and set on maximum (e.g., in Edit Method in Bruker's Paravision 5.1 or using the "Instruction" tab (GOP) and "Setup" tab in Paravision 6).
4. Start the SINGLEPULSE sequence using setup mode. If the 19 F spectral signal within the acquisition-window is too low, add more averages until a signal is clearly visible. Adjust the basic frequency in order to center the 19 F spectral peak at 0 Hz in the acquisition window. Apply this basic frequency, press Stop and apply.
5. Setup a TurboRARE 3D protocol for the 19 F scans.
6. Use the same geometry used for anatomical 1 H imaging but reduce the matrix size for increased SNR and use a rare factor of at least 32. Set nucleus to 19 F. Deselect automatic adjustments (as above). For an example of parameters, please see Note 7.
7. When the scans are finished retract the mouse-holder from the MR scanner. Disconnect the mouse carefully from the holder. If the mouse is not sacrificed for ex vivo analysis (e.g., high resolution 19 F MRI of the kidney, see below) following the MR scans, closely monitor until it has completely recovered from anesthesia. Body temperature regulation might still be affected after the anesthesia, so during the recovery process, put the mouse in a separate cage that is placed on a warm temperature regulated pad. Once the mouse has completely recovered from anesthesia, you may return it to its holding cage and to the animal room.

High Spatial Resolution 19 F MRI of Ex Vivo Kidney Using a 19 F CryoProbe
Kidney inflammation can also be studied with high spatial resolution ex vivo 19 F MRI, for example, by using a transceive 19 F CryoProbe, which we previously used to study brain inflammation in a model of CNS autoimmunity [25]. An example of a high resolved ex vivo 1 H/ 19 F MRI of the kidney is shown in Fig. 2. 1. At the end of the in vivo experiment, anesthetize mice with a terminal dose of ketamine and xylazine. Ensure the required level of anesthesia is reached (no response following toe pinch).
2. Transcardially perfuse mouse with 20 ml PBS followed by 20 ml 4% paraformaldehyde.  2. The FLASH_3D protocol is especially adapted for whole-body imaging of mice (the gradient-system and the volumeresonator need to have a linear region of about 8 cm).
3. The block pulse program (SINGLEPULSE) contains the necessary elements for a simple transmit/receive experiment, sending a pulse and acquiring an FID afterward. 4. A 2D version of this sequence is also available, which allows thicker slices for a general overview but suffers from low SNR for most in vivo applications.
5. Special attention should be given when studying inflammation in models where tissue oxygen levels are likely to change, for example, following ischemic events. In these cases, T 1 weighting needs to be reduced at cost of SNR efficiency by increasing TR ) T 1 (typically TR ¼ 3-5 Â T 1 ). 8. The polydispersity index (PdI) is extrapolated from the DLS function and quantitatively describes the particle size distribution best. PdI ranges from 0.01 for monodispersed particles to 0.7 for particles that have a very broad size distribution. The zaverage diameter gives the mean diameter based on intensity of scattered light and sensitive to presence of large particles, peak diameter, peak width, and PdI. 9. In MPO-AAV mice we administered one bolus of PFCE nanoparticles intravenously (80 μmol in 100 μl) 8 weeks following transplantation of bone marrow cells.
10. Mice can be alternatively anesthetized with an intraperitoneal injection of ketamine and xylazine.
11. Shimming is particularly important, since macroscopic magnetic field inhomogeneities affect the exact resonance frequency of the PFCE compounds and might affect quantification of the 19  Also, our research is funded by the Deutsche Forschungsgemeinschaft to SW (DFG WA2804), AP (DFG PO1869), and AS (DFG SCHR7718). This chapter is based upon work from COST Action PARENCH IMA, supported by European Cooperation in Science and Technology (COST). COST (www.cost.eu) is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable scientists to enrich their ideas by sharing them with their peers. This boosts their research, career, and innovation. PARENCHIMA (renalmri.org) is a community-driven Action in the COST program of the European Union, which unites more than 200 experts in renal MRI from 30 countries with the aim to improve the reproducibility and standardization of renal MRI biomarkers.