In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Summary

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Abstract

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

Introduction

Inflammation is a highly regulated biological response involved in a variety of human diseases, including microbial infection ¹, wound healing ², diabetes ³, cancer ⁴, cardiovascular ⁵, neurodegenerative ⁶, and autoimmune diseases ⁷. Tissue inflammation requires proper coordination of various immune cells in order to achieve pathogen clearance, tissue repair, and disease resolution. Neutrophils and macrophages are key immune mediators of tissue inflammation. In the acute phase of inflammation, neutrophils are the first responders to various harmful stimuli and tissue damage ⁸. The ....

Protocol

Note: All animal studies were performed under approved institutional protocols and animal care guidelines.

1. Reagents and Solutions

1. PMA solution for s.c. inoculation: Prepare a stock solution of phorbol 12-myristate 13-acetate (PMA) at 5 mg/ml in DMSO. Store the stock solution at -20 °C. Before inoculation, thaw the stock solution and dilute to 1 mg/ml PMA in PBS.
2. LPS solution for s.c. inoculation: Dissolve lipopolysaccharide (LPS from Salmonella enterica serotype enteritidis) in sterile PBS at 1 mg/ml prior to s.c. inoculation.
3. Luminol solution for acute phase imaging: Dissolve....

Results

We performed longitudinal bioluminescence imaging to assess acute and chronic inflammation in experimental inflammation models. Phorbol 12-myristate 13-acetate (PMA) is a potent protein kinase C (PKC) agonist that activates Phox for superoxide anion production and triggers intense acute inflammatory responses ²¹. S.C. injection of 50 mg of PMA in NCr nude mice caused rapid skin irritation and acute inflammation at injection sites ^{18, 22, 23}. We performed daily imaging for 4 days with the first image.......

Discussion

In this report, we demonstrate a bioluminescence method for non-invasive imaging of inflammation in living animals. Taking advantage of two luminescent substrates, luminol and lucigenin, the method can distinguish different phases of inflammation. Luminol bioluminescence is associated with neutrophils in the acute phase of inflammation, whereas lucigenin bioluminescence is mediated by macrophages in the chronic phase. Relatively small (M.W. = 177.16 g/mol) and electrically uncharged, luminol can readily penetrate both pl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phorbol 12-myristate 13-acetate (PMA)	Sigma-Aldrich Co., St. Louis, MO	P8139
Lipopolysaccharide from Salmonella enterica serotype enteritidis	Sigma-Aldrich Co., St. Louis, MO	L2012
Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt)	Sigma-Aldrich Co., St. Louis, MO	A4685
Lucigenin (bis-N-methylacridinium nitrate)	Sigma-Aldrich Co., St. Louis, MO	M8010
IVIS Spectrum imaging system with Living Imaging 4.2 software package	Caliper LS/Perkin Elmer, Hopkinton, MA