Rapid quantification of inflammation in tissue samples using perfluorocarbon emulsion and fluorine-19 nuclear magnetic resonance

Abstract

Quantification of inflammation in tissue samples can be a time-intensive bottleneck in therapeutic discovery and preclinical endeavors. We describe a versatile and rapid approach to quantitatively assay macrophage burden in intact tissue samples. Perfluorocarbon (PFC) emulsion is injected intravenously, and the emulsion droplets are effectively taken up by monocytes and macrophages. These 'in situ' labeled cells participate in inflammatory events in vivo resulting in PFC accumulation at inflammatory loci. Necropsied tissues or intact organs are subjected to conventional fluorine-19 ((19)F) NMR spectroscopy to quantify the total fluorine content per sample, proportional to the macrophage burden. We applied these methods to a rat model of experimental allergic encephalomyelitis (EAE) exhibiting extensive inflammation and demyelination in the central nervous system (CNS), particularly in the spinal cord. In a cohort of EAE rats, we used (19)F NMR to derive an inflammation index (IFI) in intact CNS tissues. Immunohistochemistry was used to confirm intracellular colocalization of the PFC droplets within CNS CD68+ cells having macrophage morphology. The IFI linearly correlated to mRNA levels of CD68 via real-time PCR analysis. This (19)F NMR approach can accelerate tissue analysis by at least an order of magnitude compared with histological approaches.

Figure 1. Overview of inflammation quantification of intact tissue samples using ¹⁹F NMR

PFC emulsion is injected i.v. and is taken up by monocytes and macrophages. These labeled cells participate in inflammatory events in vivo resulting in an accumulation of ¹⁹F at inflammatory loci. Conventional ¹⁹F NMR spectroscopy of panels of intact tissue samples is used to measure histogram profiles of the inflammatory index (IFI), proportional to tissue macrophage burden.

Figure 2. NMR results in the EAE (stage 2) and control rats

(A) ¹⁹F NMR spectrum of a fixed, intact cervical spinal cord segment from EAE rat. The PFC displays a single peak at −92 ppm and the TFA reference peak is at −76 ppm. (B) Average inflammation index (IFI) along spinal cord axis in EAE (n = 8) and control (n = 3) rats (closed and open circles, respectively). CRBL, cerebellum; BS, brainstem. The IFI is expressed as the number of ¹⁹F atoms per gram of tissue. (C) ¹⁹F NMR measurements of organ and tissue biodistribution of the PFC emulsion in a control rat shows RES uptake.

Figure 3. Histological sections of cervical EAE (stage 2) and control spinal cords

(A) H&E stained section (10×) in EAE (left panel) and control (right panel) spinal cords, where the top and bottom panels are dorsal and ventral white matter, respectively. EAE pathology (asterisks) shows significant perivascular infiltrates (blue) compared with control. (B) Fluorescent images (40×) of immunostained white matter regions showing punctate deposits of PFC-DiI (red) within CD68+ cells (green). Data show that PFC droplets colocalized within CD68+ cells that have morphologies consistent with monocytes and macrophages. (C) Fluorescent micrograph (40×) showing an absence of DiI-PFC (red) colocalization in GFAP-stained cells (green).

Figure 4. Quantitative analysis of macrophage burden in EAE spinal cord tissue using real-time PCR in a single rat

(A) Correlation between IFI, obtained from ¹⁹F NMR, and CD68 mRNA levels. Data shows a linear correlation (R = 0.89), consistent with the PFC internalization into macrophage. The mRNA levels are normalized to gapdh. (B) Plot of IFI versus GFAP mRNA shows no correlation (R = −0.13), suggesting that PFC is not taken up by astrocytes.