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Rapid quantification of inflammation in tissue samples using perfluorocarbon emulsion and fluorine-19 nuclear magnetic resonance
 
Eric T. Ahrens, Won-Bin Young, Hongyan Xu, and Lisa K. Pusateri
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA
BioTechniques, Vol. 50, No. 4, April 2011, pp. 229–234
Full Text (PDF)
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 (19F) 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 19F 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 19F NMR approach can accelerate tissue analysis by at least an order of magnitude compared with histological approaches.

Rapid, quantitative scoring of inflammation in tissue specimens is a common need in many facets of biomedical research. Discovery and preclinical studies often rely on histological processing and analysis of a panel of tissues obtained from animal models, and these procedures are often viewed as time-consuming and expensive, and a bottleneck in research and development endeavors. Here, we describe a platform to rapidly and quantitatively assay macrophage infiltration in intact tissue samples. In this approach, we utilize a perfluorocarbon (PFC) emulsion reagent that labels phagocytic monocytes and macrophages in vivo, and conventional fluorine-19 (19F) nuclear magnetic resonance (NMR) spectroscopy is used as a quantitative readout of inflammation in intact, excised tissue samples. The approach requires no tissue preparation other than an optional fixation step.

Assaying inflammation often involves tissue-destructive methods. Commonly, thin-sectioning (4–10 ┬Ám) of embedded tissues is used in combination with one of more stains, for example, using colorimetric hematoxylin and eosin (H&E), or using immunoreactive reagents that highlight cell-specific markers or molecular events. Organ analysis of inflammation often requires serial sectioning and the preparation of a large number of slides. Periodic sampling of serial sections for the sake of accelerating analysis may lead to bias or quantification inaccuracies, or may necessitate a large experimental group to achieve statistical significance. Thus, widespread biodistribution analysis of a large panel of tissues taken from a single animal—much less an experimental group—can be a laborious undertaking. Although great strides have been made to increase tissue histology throughput, section preparation and analysis can only be made semiautomated at this time.

Alternatively, ‘bulk’ cellular, biochemical, or molecular analyses of disrupted tissue samples have been developed to assay inflammation. For example, approaches include monitoring the expression levels of selected biomarkers in peripheral blood mononuclear cells using flow cytometry (1), mRNA profiling using real-time PCR, or ELISA to detect monocyte-specific markers or pro-inflammatory cytokines (2,3).

In this article, we describe an alternative approach to assay inflammation (Figure 1), without the need for tissue disruption, thereby potentially accelerating studies. The approach uses a PFC emulsion reagent comprised of a colloidal suspension of non-toxic PFC formulated into small (~150-nm diameter) emulsion droplets. Following i.v. injection, the droplets are taken up by phagocytic cells (4-7), predominately monocytes and macrophages, and to a small degree neutrophils. These ‘in situ’ labeled cells subsequently participate in inflammatory events in vivo, resulting in an accumulation of PFC at inflammatory loci. Necropsied tissues samples, or intact organs, are subjected to conventional 19F NMR analysis to quantitatively measure the total fluorine content per sample, which is proportional to the inflammatory burden. There is negligible natural abundance of 19F in soft tissues, thus this approach offers high specificity to inflammation.



PFC-based reagents are biologically safe, even at very high doses. PFC emulsions have been studied clinically for many years as potential artificial blood substitutes in humans (8,9), although the PFC molecule used in the present study is different than those used for blood substitutes and has been optimized for 19F NMR-MRI applications. Numerous in vitro studies have shown that intracellular labeling with PFC does not affect cell phenotype and function (10-14).

As an example of the applicability of these methods, we deliver PFC emulsion reagent intravenously to the dark agouti (DA) rat model of experimental allergic encephalomyelitis (EAE) (15), a widely used model for human multiple sclerosis (MS). EAE has many clinical and histopathological similarities to MS (16). EAE is most commonly induced by immunizing animals with myelin proteins or their disease-inducing peptides (16). Clinical disease develops when primed CD4+ T cells enter the central nervous system (CNS) and recognize their cognate self-antigen presented in the context of MHC class II molecules. The resulting perivascular and parenchymal infiltrations in the CNS, particularly in the spinal cord, include a large number of macrophages and eventually lead to demyelination and clinical paralysis. In the EAE model, the ability to rapidly assay the inflammatory biodistribution in the CNS would be tremendously beneficial in attempts to understand the pathogenesis of autoimmunity and to design therapeutic interventions. Overall, the methods described herein can dramatically reduce the time to evaluate macrophage involvement in a wide variety of acute and chronic inflammatory models.

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