Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

Summary

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Abstract

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.

Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Introduction

Autoimmune diseases such as rheumatoid arthritis (RA) or psoriasis vulgaris are graded as delayed-type hypersensitivity reactions (DTHRs).¹ RA is a common autoimmune disease characterized by erosive synovitis and joint destruction.² Inflamed arthritic joints demonstrate infiltration and proliferation of inflammatory cells, an increased expression of pro-inflammatory cells leading to pannus formation, cartilage and bone destructions.^3,4 The cleavage of extracellular matrix molecules, such as collagen by matrix metalloproteinases (MMPs), is essential for tissue con....

Protocol

All procedures described in this paper, followed the guidelines and international standards of the care and use of laboratory animals and were approved by the local Animal Welfare and Ethics Committee of the Country Commission Tuebingen, Germany. 8 - 12 weeks old BALB/c and C57BL/6 mice were kept on a 12 h:12 h light:dark cycle and were housed in IVCs and standardized environmental conditions at 22 ± 1 °C in groups of 2 - 5 with water and food access ad libitum.

1. Material Preparation

1. Dilute the OI dye for near infrared fluorescence imaging according to the respective data sheet directly before injection. The....

Results

To induce rheumatoid arthritis (RA) in naïve BALB/c mice, animals were injected i.p. with auto-antibodies (1:1 dilution with 1x PBS) against GPI on day 0. The maximum inflammation (ankle swelling) in this GPI-serum induced RA model is on day 6 post injection¹¹. Therefore, 2 nmol of the activatable OI dye was prepared and injected i.v. in the tail vein of arthritic mice and healthy control animals on day 5. 24 h after injection (day 6), mice were a.......

Discussion

OI is a very useful, fast and inexpensive tool for non-invasive in vivo molecular imaging in preclinical research. A particular strength of OI is the capability to monitor highly dynamic processes like inflammatory responses. Moreover, OI allows one to follow the course of a disease for an extended period of time, ranging from days to weeks.

OI has several advantages over other in vivo imaging modalities such as positron-emission tomography (PET) or magnetic resonance imaging.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cornergel	Gerhard Mann GmbH	1224635	ophthalmic ointment
Forene	Abbott GmbH	4831850	isoflurane
U40 insulin syringe	Becton Dickinson and Company	324876
Heparin	Sintetica	6093089
High-Med-PE 0.28 x 0.61 mm	Reichelt Chemietechnik GmbH+Co	28460	polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61 mm
BD Regular Bevel Needles, 30 G	Becton Dickinson & Co. Ltd.	305106	30 G injection cannula
RTA-0011 isoflurane vaporizer	Vetland Medical Sales and Services LLC	-
Artagain drawing paper	Strathmore Artist Paper	446-8	coal black
IVIS Spectrum	Perkin Elmer	124262	Optical imaging system
BD Regular Bevel Needles, 25 G	Becton Dickinson and Company	305122
2-Chloro-1,3,5-trinitrobenzene	Sigma Aldrich GmbH	7987456F	TNCB
MMPSense 680	Perkin Elmer	NEV10126	fluorescent imaging dye
Oditest	Koreplin GmbH	C1X018	mechanical measurment
Miglyol 812	SASOL	-	Oil
BALB/C, C57BL/6	Charles River Laboratories	-	Mice used for experiements
PBS	Sigma Aldrich GmbH		For dilution of the RA serum
Pipette (100 µL)	Eppendorf		Used for TNCB application
shaver	Wahl	9962	Animal hair trimmer
Living Image	Perkin Elmer		Imaging software to measure OI