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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 conversion and angiogenesis and causes tissue destructions.^5,6 Contact hypersensitivity reactions (CHR) are characterized by aggregation of neutrophils leading to an oxidative burst.⁷ Similar to RA, MMPs in CHR are involved in tissue conversion, cell migration and angiogenesis in order to establish chronic inflammation.

To investigate RA, the glucose-6-phosphate isomerase (GPI)-serum injection mouse model was used.⁸ Serum from transgenic K/BxN mice containing antibodies against GPI, was injected into naïve BALB/c mice after which rheumatic inflammation began to develop within 24 h with a maximum of ankle swelling on day 6 after GPI-serum injection (see 1.1). To analyze chronic CHR, C57BL/6 mice were sensitized with trinitrochlorobenzene (TNCB) on the abdomen. The right ear was challenged up to 5 times starting 1 week after sensitization (see also 1.1 and 1.2).

Noninvasive small animal OI is a technique based on the in vivo investigation of fluorescent-, chemiluminescent- and bioluminescent-signals, which are mainly used in preclinical research. The acquired semi-quantitative data gives insights into the molecular mechanisms in the organs and tissues of healthy as well as diseased experimental animal models, and enables longitudinal follow up measurements (e.g. to assess therapeutic response profiles in vivo). A big advantage of longitudinal studies is the reduction of animal numbers, as the same animals can be measured in follow up studies at several time points instead of using different mice per time point. The resolution of OI allows detailed functional imaging of organs and even smaller tissue structures in experimental animals.

The use of specific excitation and emission filters with a narrow transmission spectrum, a protection against scattered light by a lightproof "dark box" and a sensitive charged-coupled device (CCD) camera, which is cooled in many devices down to -70 °C, allows highly specific and sensitive measurements of fluorescence signals.

By using fluorescent agents with excitation- and emission-spectra in the near-infrared fluorescence window (650 - 950 nm), signal-to-noise ratios can be improved significantly. The near-infrared fluorescence window is characterized by a relatively low absorption of the signal by hemoglobin and water as well as a low background auto-fluorescence.⁹ This allows a penetration depth of up to 2 cm in the tissue of small animals. OI-probes can address a target directly (e.g. by a fluorescence labeled antibody) or can be activated in the target tissue (e.g. by proteases). Activatable OI probes are optically silent in their inactivated form due to the Förster resonance energy transfer (FRET) to a quenching moiety, which transfers the excitation energy within the molecule to another domain. If the dye is cleaved (by a protease for example) the energy is no longer transferred within the molecule and a fluorescent signal can be detected by OI. This allows the design of OI probes with high specificity for distinct biological processes and excellent signal-to-noise-ratios.

The following protocol explains in detail the preparation of the animals, the OI measurements using an Activatable OI probe to image MMP-2, -3, -9 and -13 activity in vivo and two experimental models of inflammation (RA, CHR).

Protokół

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 activatable OI dye (commercial probe with an excitation at 680 nm) to measure MMPs in vivo is ready to use at a concentration of 20 nmol in 1.5 mL 1x PBS. Gently shake or vortex the solution before use. OI dye can be stored at 2 - 8 °C for up to 6 months when protected from light.
2. For intravenous (i.v.) injections prepare a venous catheter. Use a 20 U (0.5 mL) insulin syringe filled with 0.9% saline solution (containing 10 injection units of heparin in 50 mL), and a 30-gauge needle attached to a polyethylene catheter. Inject the recommended dose of 2 nmol per mouse of the OI dye.

2. Induction of Rheumatoid Arthritis and Chronic Contact Hypersensitivity Reaction

1. Rheumatoid arthritis:
   1. Dilute 100 µL of serum (gained from K/BxN mice¹⁰) containing antibodies (AB) against glucose-6-phosphate isomerase (GPI) 1:1 with 1x PBS (200 µL) to induce RA. Store the diluted serum at -80 °C. Detailed procedures for induction of RA are described by Monach et al.⁸.
   2. To induce RA, lift each mouse gently by its tail and inject 200 µL of the diluted serum intraperitoneal (i.p.) on day 0 of the experiment. After the injection place the mouse directly back into its cage.
   3. Optionally, measure the ankle diameters of each ankle, before AB injections and define an "arthritic score" (Figure 1A). Continue measurement of ankle swelling daily until day 6 after GPI-serum using a mechanical measuring device (micrometer).
   4. Inject the activatable OI dye (step 1.1 - 1.2), to measure in vivo MMP activity i.v. into the tail vein of RA mice on day 5 after GPI-serum injection and into the tail vein of control mice. Perform optical imaging experiments 24 h after tail vein injection.
2. Contact hypersensitivity reaction:
   1. For sensitization of C57BL/6 mice, prepare a 5% TNCB solution dissolved in a 4:1 mixture of acetone/oil.
   2. Anaesthetize C57BL/6 mice using 1.5% isoflurane vaporized in 100% oxygen (1.5 L/min). Once the mice are anesthetized, place it in a self-made nose cone (a cut 5 mL polyethylene syringe, connected to the 1.5 vol% isoflurane tube) and maintain anesthesia. Apply vet eye ointment on anesthetized animals to prevent dryness of the eyes while anesthetized.
   3. Shave the abdomen carefully (2 x 2 cm) using a small animal hair trimmer. Avoid injuring the skin, as this can lead to unspecific fluorescent signals within the animal and can influence the results of the study.
   4. Apply 80 µL of the 5% TNCB solution slowly at the shaved abdomen area using a 100 µL pipette to sensitize the animal. Place the mouse gently back into its cage taking care to ensure that the mouse’s eyes do not come in contact with the bedding to avoid corneal injury and avoid low body temperatures during recovery phase.
   5. Six days after sensitization, prepare a 1% TNCB solution (dissolved in a 9:1 mixture of acetone/oil) and apply 20 µL at the right ear using a pipette to elicit acute and chronic CHSR.¹
      1. Start with the 1^st challenge on both sides of the right ear with 20 µL of 1% TNCB solution using a 100 µL pipette and repeat the challenge every second day up to five times (day 15 after sensitization) to elicit CHR (Figure 1B). Measure ear thickness daily, using a micrometer measuring device.
   6. Inject the activatable OI dye (a commercial probe with excitation at 680 nm) (step 1.1-1.2) to measure MMP activity in vivo in CHR mice 12 h after the challenge. Perform optical imaging experiments 24 h post tail vein injection (i.v.).

3. Animal Preparation for Optical Imaging

1. Switch mouse chow (at least 3 days before imaging) to low or non-fluorescence chow (e.g., manganese free) to avoid the interference of auto-fluorescence (around 700 nm) with the fluorescence signal of the used OI agents.
2. Place the animal into an anesthesia box and anesthetize using 1.5 vol% isoflurane vaporized with oxygen (1.5 L/min) (oxygen can be replaced by air but needs to be standardized within one experiment).
3. When the mouse is visibly anesthetized, place it in a self-made nose cone (build by a cut 5 mL polyethylene syringe, connected to the 1.5 vol% isoflurane tube) and maintain anesthesia.
4. Shave the animal carefully on the target site (2 cm x 2 cm) using a small animal hair trimmer. Avoid injuring the skin, as this can lead to unspecific fluorescent signals within the animal and can influence the results of the study.
   NOTE: Hairs can absorb the fluorescence signal during OI (dependent on the mouse strain, more or less absorption is observed) depending on the region of interest (ROI).
5. For i.v. injection, place the tail of the mouse in warmed water, to induce vasodilation, gently cleanse the skin at the injection site with alcohol, and start by placing the catheter at the distal site of the tail. Place the "cut" edge of the needle in, at an angle of 20° into the tail vein and test the correct placing of the catheter by re-suspending the syringe.
6. If the catheter is placed correctly, replace the syringe and inject the OI probe (2 nmol). After injection, replace the syringe and inject 25 µL of 0.9% saline solution to fully clear the dead volume of the polyethylene tube.
   NOTE: The tissue half-life time of the used OI dye (a commercial probe with excitation at 680 nm) for measuring MMP activity in vivo is 72 h. To ensure a complete clearance, a re-injection of the dye is not recommended earlier than 7 days after a prior injection.

4. Optical Imaging

1. Place a black plastic or paper sheet in the black box of the OI-scanner in the center of the field of view (FOV).
2. Set up a measuring protocol and choose the right wavelength (excitation: 680 ± 10 nm and emission: 700 ± 10 nm) and imaging parameters.
   NOTE: In some OI systems, the setup for several imaging dyes is pre-defined.
3. To choose the correct protocol for fluorescence imaging, open the imaging software (provided by the manufacturer) and initialize the system. Most CCD cameras need to cool down to their working temperature, and this can take 10 minutes. For reliable results, wait until the system is ready.
4. Observe the in vivo imaging system acquisition control panel pop up and each filter pair selected will represent one image in the sequence. In this case, acquire one image with the filter pairs for the commercial dye with and an excitation and emission wavelength of 680 ± 10 nm and 700 ± 10 nm, respectively, and start the measurement (Press "Acquire sequence"). For more detailed instructions, please see the manual of the manufacturer.
5. Label the images appropriately and save the information in the "Edit Image Labels" window, which will pop up following the "Acquire sequence".
6. Take a baseline scan of each animal before the injection of the OI dye, or use naïve control animals to differentiate the background signal.
7. h after the tail vein injection of the OI dye, place the animals in the center of the FOV, in a position to measure the highest signal in the OI system, and start the measurements.
   NOTE: Important: Hypothermia can significantly influence the distribution of imaging agents. Make sure that the stage is heated up to 37 °C to avoid hypothermia of the animal. Imaging can be performed simultaneously with animals in groups of 1 to 5 mice. Choose the size of the FOV depending on the number of animals to measure at the same time. You can take a bright field image to check whether each mouse is visible.

5. Data Analysis

NOTE: Perform data analysis using the image software following manufacturer's protocol.

1. For measurements of RA, use the calibrated unit of the photon emission as shown in Figure 2, whereas chronic CHSR are pictured as signal intensity in percent (efficiency).
2. To draw ROIs manually, use the tool plate. For the analysis of the RA images use a standardized circle, placed around the highest signal in all ankles and paws of each animal. To analyze the CHSR, place the ROIs around the whole right and left ear according to the bright field image.
3. To measure photon emission or signal intensity values in the specific drawn ROI, press "measure". The system will provide values in the drawn ROI for descriptive statistical analysis.

Wyniki

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 w...

Dyskusje

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...

Materiały

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