Fluorine (¹⁹F) MRI to Measure Renal Oxygen Tension and Blood Volume: Experimental Protocol

Abstract

Fluorinated compounds feature favorable toxicity profile and can be used as a contrast agent for magnetic resonance imaging and spectroscopy. Fluorine nucleus from fluorinated compounds exhibit well-known advantages of being a high signal nucleus with a natural abundance of its stable isotope, a convenient gyromagnetic ratio close to that of protons, and a unique spectral signature with no detectable background at clinical field strengths. Perfluorocarbon core nanoparticles (PFC NP) are a class of clinically approved emulsion agents recently applied in vivo for ligand-targeted molecular imaging. The objective of this chapter is to outline a multinuclear ¹H/¹⁹F MRI protocol for functional kidney imaging in rodents for mapping of renal blood volume and oxygenation (pO₂) in renal disease models.

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 a separate chapter describing the basic concept of functional imaging using fluorine (¹⁹F) MR methods.

1 Introduction

In healthy kidneys, the countercurrent design of blood vessels and tubules maintains the necessary osmolar gradient to permit efficient urinary concentration, yet the system operates under conditions of a relatively hypoxic environment in some regions that requires extensive extraction of oxygen from flowing blood [1, 2]. During acute and chronic kidney injury, ischemia challenges effective O₂ delivery to the capillary beds of the renal tubules where extraction of extra oxygen is marginal, resulting in heightened susceptibility to ischemic damage that may culminate in renal failure [3]. Besides the traditional biochemical metrics for assessing renal failure (e.g., blood urea nitrogen, creatinine), ¹H/¹⁹F imaging offers a multiparametric quantifiable approach to functional imaging in kidney injury that avoids the use of potentially nephrotoxic gadolinium (Gd) based contrast agents [3].

Perfluorocarbon core nanoparticles (PFC NP) are a class of emulsion agents clinically approved as blood substitutes that have been applied recently for in vivo for targeted molecular imaging and noninvasive oxygen tension (pO₂) assessment [1,2,3]. As compared with standard iodinated or Gd contrast agents, PFC NP are biologically inert and exhibit a good safety profile with no renal toxicity reported in both animals and human [3]. Because PFC NP (nominal size 200–250 nm) are not cleared through glomerular filtration, they do not increase kidney workload, which is a major risk factor for kidney failure with the use of other agents that undergo renal clearance [3]. For imaging purposes, the unique ¹⁹F MR signal emanating from the fluorine core of PFC NP can be measured directly in vivo in quite sparse concentrations (e.g., picomolar voxel amounts) with no background signal, as contrasted with other paramagnetic or superparamagnetic moieties that exert indirect contrast effects on circulating protons [4,5,6]. PFC NP have been demonstrated as effective functional probes for regional blood volume as the detected ¹⁹F signal intensity directly reflects the actual quantity of PFC NP within a voxel. Furthermore, for pO₂ measurements, the ¹⁹F longitudinal relaxation rate (R₁) responds directly to local oxygen content in a linear manner [1, 2].

Herein we describe ¹H/¹⁹F MRI for monitoring of renal perfusion in the kidney of rodents in a step-by-step experimental protocol. The rationale for choosing acquisition parameters is described in generic terms, together with specific parameter examples.

This experimental protocol chapter is complemented by a separate chapter describing the basic concept of functional imaging using fluorine (19F) MR methods, which is 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.

2 Materials

2.1 Animals

This experimental protocol is applicable to mice and rats.

2.2 Lab Equipment

Enumerated below are the contrast agents and equipment components needed:

• 1. Perfluorocarbon Nanoparticle Preparation: PFC NP (~200–250 nm) can be formulated with a variety of PFC that are liquid at body temperature. Here we describe either perfluorooctylbromide (PFOB) or perfluoro-15-crown-5-ether (CE). The PFOB or CE emulsions are composed of 20% or 40% (v/v) of PFOB or CE, 2.0% (w/v) of a surfactant commixture, and 1.7% (w/v) glycerin in water (see Note 1).

• 2. Gases: O₂, N₂, and compressed air, as well as a gas-mixing system to achieve required changes in the oxygen fraction of inspired gas mixture (FiO₂). In addition to room air, the following gas mixtures are required during the experiment: 10% O₂–90% N₂ for hypoxia and 100% O₂ for hyperoxia.

• 3. Anesthesia: An MRI-compatible slow infusion system is needed for anesthesia with ketamine–xylazine (e.g., syringe pump). Isoflurane is not recommended for ¹⁹F imaging as it could interfere with the detected ¹⁹F MRI signal from PFC NP.

• 4. Small Animal Monitoring System: An MRI-compatible small animal physiological monitoring system (e.g., SAI Inc., USA) is needed for real time respiratory monitoring and respiratory triggering and/or gating. Respiratory gating is recommended during ¹⁹F MRI acquisition because it reduces motion artifact and preserves ¹⁹F signal intensity in the final images, which could otherwise be degraded by motion.

• 5. Small Animal Holder: a dedicated customer built small animal holder is needed to accommodate animal imaging and for maintenance of body temperature at 37 °C. A warm water blanket or warm air blower can be used for maintaining body temperature of animals (see Note 2).

• 6. Animal Surgery Suite: A fully equipped animal surgery suite is needed for preparing animal models of renal injury (see Note 3).

2.3 MRI Hardware

The technique described in this chapter is tailored for an 11.7 T MR system (e.g., Varian, USA) but advice for adaptation to other field strengths is given where necessary. In general, the MRI scanner should be equipped with a wide band spectrometer and a wide band RF power amplifier. The scanner needs to be functional at both ¹H and ¹⁹F frequencies.

• 1. ¹H/¹⁹F dual-tuned Coils: use actively decoupled coil pair for renal imaging applications. Such coils can be custom-built as described in our prior publications [7] or obtained from commercial coil manufacturers (see Note 4).

• 2. Transmit Coils: use a volume transmit coil to generate a homogeneous B₁ field that covers both kidneys. Tune and match to the ¹⁹F frequency.

• 3. Receive Coils: Place a surface receive coil on the dorsal side of the animal to achieve high sensitivity to ¹⁹F signal from the two kidneys. Tune and match to the ¹⁹F frequency.

2.4 MRI Techniques

• 1. ¹H MRI: a gradient echo sequence is employed to locate two imaging slices with 2 mm thickness, positioned 1–2 mm apart, and centered respectively at the left and right kidney.

• 2. ¹H MRI: a multiecho gradient-echo blood oxygenation level dependent (BOLD ) scan can be performed. For an example of parameters used for a specific setting (see Note 5).

• 3. ¹⁹F MRI: For spin density ¹⁹F MRI, TR is set to 4 s. For T₁-weighted MRI, TR is set to two respiration periods, or ~300 ms. All other imaging parameters are identical in these two sets of ¹⁹F MRI (see Note 6)

• 4. ¹⁹F MRI Oxygenation Measurement: For PFC NP dependent oxygenation measurements in fast flowing blood of major blood vessels, a Blood Flow-Enhanced-Saturation-Recovery (BESR) sequence [8] can be utilized to minimize in-flow effects on the image (see Note 7).

3 Methods

3.1 PFC Nanoparticle Formulation

• 1. Prepare PFC NP emulsifying 40% (v/v) of CE (Exfluor Research Corp, Round Rock, TX), 2.0% (w/v) of a surfactant commixture, and 1.7% (w/v) glycerin, in distilled water using a microfluidic apparatus (e.g., benchtop LV1 fluidizer, Microfluidics Corporation, Westwood, MA) (see Note 8).

• 2. Determine size and charge with a submicron particle analyzer (e.g., Zetasizer, Malvern, UK) [9, 10].

3.2 PFC NP Injection and Imaging

• 1. Inject PFC NP for functional perfusion imaging to quantify blood volume and oxygenation.

• 2. Prepare an external reference for ¹⁹F signal quantification. Place the external reference close to the animal within the field of view (see Note 9).

• 3. Perform ¹H and ¹⁹F imaging 10 min to 1 h after NP injection. A shorter circulation time is preferred to maximize the intravascular ¹⁹F signal (see Note 10).

3.3 Coil Sensitivity Profiling for Quantification of ¹⁹F Signal Intensity

• 1. Acquire an RF coil sensitivity map using a saline phantom positioned at the selected imaging location to correct for the signal bias created by surface coil receive profile inhomogeneity. The saline phantom is prepared to cover the entire region of imaging FOV (see Note 11).

• 2. Compensate for the spatial inhomogeneity of the RF coil sensitivity profile by multiplying the correction factor obtained from the phantom image (see Note 12) with the in vivo measured image signal intensity at the corresponding locations.

3.4 Calibration of ¹⁹F R₁ vs. pO₂ Curve In Vitro

• 1. Perform an in vitro calibration of ¹⁹F relaxation time (R₁) as a function of pO₂ using at least three different O₂ concentrations (e.g., 0%, 21%, 100% O₂ balanced with N₂) by bubbling gas mixtures into a PFC emulsion sample for 30 min at 37 °C (Fig. 1).

• 2. Perform ¹⁹F T₁ measurements with inversion recovery spectroscopy at TR > 10 s, and at least ten different inversion delays (T_I) ranging from 3 ms to 5 s. Use exponential fitting to extract R₁ from T₁ measurements and linear fitting to calibrate the relationship between ¹⁹F R₁ and pO₂.

Fig. 1

In vitro calibration shows the ¹⁹F R₁ (=1/T₁) of PFC NP as a function of pO₂. Data were collected at 11.7 T using a PFC NP phantom. Temperature of the PFC NP phantom was maintained at 37 °C. The correlation coefficient R > 0.99 (From Hu et al. [1])

3.5 Quantification of ¹⁹F Based pO₂ Measurement In Vivo

• 1. Create two sets of images, one using a short TR and one using a long TR, for T₁ mapping (see Note 13).

• 2. Determine the actual TR using the external ¹⁹F standard (see Note 14).

• 3. After determining the actual short TR, perform voxel-wise T₁ mapping of the kidney using the standard saturation recovery signal model:

  $$ S={S}_0\left(1-{e}^{\frac{TR}{T_1}}\right). $$

  (1)

• 4. Translate T₁ values into corresponding pO₂ levels according to the predetermined calibration curve.

4 Notes

• 1. The CE signal intensity is effectively greater for narrow band reception as all fluorine atoms contribute to a single spectral peak, as compared to PFOB where chemical shifts apportion the signal into several peaks (Fig. 2). Suppliers of PFC include Exfluor Research Corporation (Round Rock, TX). The clearance half-life for PFOB is ~3–5 h depending on species and whether the PFC contrast material is molecularly targeted or not [11]. The CE materials exhibit a longer tissue residence time in bloodstream than PFOB compounds, which makes them problematic for clinical use or repeat studies. For the present protocol, no targeting ligands are attached to the surface of the PFC NP to avoid changing the pharmacokinetics of the contrast agent.

• 2. Potential MRI in-flow artifacts may be present on ¹H but not ¹⁹F MR images if a warm water blanket is used. Special attention should be given when choosing the animal holder and the MRI coils as reduced animal motion during lengthy MRI acquisitions will improve image quality.

• 3. In general, ischemic renal injury can be produced unilaterally or bilaterally by exposing the kidneys either through the abdomen or retroperitoneally, and then clamping the renal artery for a selected interval [1, 2]. The animal surgery suite does not need to be located in the MRI facility but should be close enough for imaging a short time after surgery and later for tissue harvest after imaging is completed if indicated.

• 4. Despite being slightly off-resonance for ¹H imaging, the ¹⁹F coil pair should be sufficient to generate high quality anatomic images within <1 min scan time. The size of the transmit and receive coils should be designed to match the animal size, so that different coil sizes will be needed for imaging mice and rats. For imaging at field strengths lower than 11.7 T, it is recommended to use double tuned receive coils that simultaneously function at ¹H and ¹⁹F frequency to assure maximal detection efficiency while avoiding potential image mismatches between ¹H and ¹⁹F images that may occur as a result of manual coil retuning.

• 5. The imaging parameters of BOLD at 4.7 T are: TR = 100 ms, TE = 1.96 ms with 1.75 ms increment for every echo, flip angle = 10°, field of view = 26 × 26 mm², voxel size = 0.2 × 0.2 × 2 mm³. Field of view and voxel size should be adjusted based on animal size to achieve optimal image quality.

• 6. ETL = 4, TE = 11.5 ms, receiver bandwidth = 20 kHz, voxel size = 0.4 × 0.4 × 2 mm³, and the field of view is identical to that for ¹H BOLD imaging.

• 7. For an example of parameters used at 4.7 T, the imaging parameters of BESR sequence are: TR = 2.5 s; TE = 2.2 ms; number of points along recovery curve = 8; number of averages = 16; in plane resolution = 1.5 mm × 0.75 mm; slice thickness = 2 mm

• 8. PFC NP can be colabeled with Alexa Fluor 594 for ex vivo fluorescence microscopy examination.

• 9. As reference, a tube of another PFC NP formulation (e.g., perfluorodecalin) at a similar PFC concentration to that administered in vivo for the imaging studies can be used.

• 10. In healthy tissues, PFC NP are generally constrained to the intravascular space because of their large particle size (200–250 nm), which ensures that the measured ¹⁹F signal intensity correlates linearly with intravascular blood volume. One has to also consider that during injury, PFC NP will escape from the circulation and will get trapped in tissue compartments. In this case the total fluorine signal would comprise both stationary (i.e., trapped in tissue) and flowing (i.e., in blood) PFC NP. Employ spin labeling [12] to eliminate the signal from trapped PFC NP, leaving only the signal from the flowing blood compartment. Apply diffusion gradients to suppress the signal from flowing blood [13] and acquire distinct signal from either compartment. For O₂ measurements, a mixture of signals could occur where trapped PFC NP exist under hypoxic conditions and the flowing PFC NP under more normoxic conditions. In this case the ¹⁹F relaxation times would represent an average of the two situations.

• 11. At 11.7 T, imaging parameters are as follows: TR = 100 ms, TE = 1.96 ms, and flip angle = 10°. For other field strengths, minimize TE and select similar TR and flip angle.

• 12. For each voxel location, the phantom image defines a correction factor matrix R(x,y,z) that is the inverse of local signal intensity I(x,y,z): R(x,y,z) = 1/I(x,y,z).

• 13. In vivo quantification of renal T₁ and pO₂ is based on the general assumption that image signal intensity in a fast spin echo sequence follows a saturation-recovery process (Fig. 3). Because short TR or T₁-weighed ¹⁹F MRI is gated with respiration, the actual short TR can vary from animal to animal.

• 14. Given that the ¹⁹F T₁ of PFC standard is 0.9–1 s at room air oxygen tension, the signal acquired with a long TR = 4 s, that is, >3 × T₁, should approximate the ¹⁹F spin-density signal of PFC NP at room temperature (Fig. 4).

Fig. 2

Chemical structure and magnetic resonance chemical shift of perfluoro-15-crown-5-ether (CE) and perfluorooctylbromide (PFOB)

Fig. 3

(a, b) Representative renal blood volume and pO₂ mapping of healthy kidneys determined by quantitative ¹⁹F MRI. (c) Representative ¹H T2* (BOLD) mapping of healthy kidneys. (d–f) Quantification of functional indexes in different anatomical regions. *P < 0.05 compared with cortex (From Hu et al. [1])

Fig. 4

Imaging of improved renal medullary perfusion after antithrombin PFC NP treatments. Here the thrombin inhibitor PPACK (phenylalanine-proline-arginine-chloromethylketone) was conjugated to the PFC NP. Mice were pretreated with PFOB PPACK NP, plain PFOB NP, or saline before ischemia induction. At 3 h after reperfusion, a single dose of CE NP was injected i.v. for ¹⁹F MRI. The ¹⁹F signal from circulating CE NP of uninjured kidney illustrates normal kidney perfusion (a). In contrast, all injured kidneys exhibited reduced ¹⁹F signal in the medulla (b–d) reflecting the severity of regional nonreperfusion. Compared to kidneys pretreated with saline or plain NP (b, c), the improved medullary perfusion in PPACK NP treated kidneys is visually appreciable (d). Using in vivo ¹⁹F MRI data, the ratio of nonperfused kidney area after reperfusion was determined. The PPACK NP treated kidneys (n = 4) developed less regional nonreperfusion than did saline (n = 6) or plain NP (n = 6) treated kidneys. *p < 0.05 compared to saline or plain NP treated kidneys. Note that the arrow in d indicates the partial overlay of ¹⁹F signal from the adjacent spleen (From Chen et al. [2])