The overall goal of this procedure is to use optical imaging to visualize an activatable probe for assessing the in vivo activity of key matrix metalloproteases, or MMPs, in experimental models of inflammation. This method can help answer key questions about underlying biological mechanism in inflammatory diseases. The main advantages of this technique are that it is fast, cheap, and easy to learn.

Moreover, a large variety of probes to target different biological processes are available. For rheumatoid arthritis induction, first use a micrometer to measure all of the ankle diameters of each animal. Next, lift the first mouse gently by the tail and intraperitoneally inject 200 microliters of diluted KBN mouse serum containing antibodies against glucose six phosphate isomerase or GPI.

Then place the animal back in its cage and measure the ankle swelling daily until day six. For contact hypersensitivity sensitization, use a small animal hair trimmer to carefully shave a two by two centimeter area of the abdomen of an anesthetized mouse and use a 100 microliter pipette to apply 80 microliters of freshly prepared 5%TNCB solution onto the exposed skin. It's very important to avoid injuring the skin at any point during the procedure, as this can lead to unspecific fluorescent signals within the animal and can impact the results of the study.

Gently place the animal back into its cage, avoiding low body temperatures during the recovery phase. Six days later, use a pipette to apply 20 microliters of 1%TNCB solution to both sides of the right ear, followed by the application of 100 microliters of 1%TNCB every second day for up to 10 days and daily measurement of the ear thickness. To measure the in vivo MMP activity, first gently vortex the activatable optical imaging dye and load a 0.5 milliliter insulin syringe with two nanomolar of the dye per mouse.

Then equip another syringe with a 30 gauge needle attached to a polyethylene catheter and place the tail of the first mouse in warm water. When the veins have dilated, insert the catheter with the cut edge of the needle at a 20 degree angle at the distal side of the tail into a dilated tail vein and re-suspend the syringe to test the correct placement of the catheter. If the catheter is appropriately positioned, replace the syringe and inject two nanomolar of the probe.

Then replace the optical imaging probe syringe with a syringe containing 0.9%saline solution and inject 25 microliters of saline into the tail vein to fully clear the dead volume of the polyethylene tube. Next, set the imaging scanner box of the optical imaging scanner to 37 degrees celsius and place up to five mice into the scanner box in the center of the field of view. 24 hours after the injection, open the imaging software and initialize the system, allowing the CCD camera to cool down to the working temperature.

When the system is ready, set the appropriate wavelengths and imaging parameters according to the florescence of the imaging probe. The in vivo imaging system acquisition control panel will pop up. Then click acquire sequence to start the measurement.

To save the acquired images, select the desired saving location. Additionally, more information can be added in the edit image labels window. Here our clearly enhanced fluorescent signal can be observed in the front paws and ankles of rheumatoid arthritis mice on day six after disease induction, compared to the minimal signal observed in control animals.

Bio-distribution analysis of the MMP activity in the organs of the experimental and control animals demonstrates a strongly enhanced signal in the ankles and forepaws exclusively in the joints of the rheumatoid arthritis mice. Interestingly, an enhanced MMP signal was also observed in both of the kidneys of the rheumatoid arthritis mice, whereas the liver and intestines demonstrate a robust MMP signal regardless of their rheumatoid arthritis status. During a chronic contact hypersensitivity reaction induced by five TNCB challenges to the right ear, a highly increased MMP activity is exhibited in the right inflamed ear compared to the left, healthy control ear.

Semi-quantitative analysis of the signal intensity of arthritic and healthy ankles or paws demonstrates a seven-fold enhanced MMP signal intensity in the arthritic ankles with a three-fold higher MMP signal intensity detected in the front paws compared to that of the healthy animals. Acute and chronic contact hypersensitivity reaction ears also exhibit a significantly increased MMP signal even after only one challenge that remains elevated through the fifth challenge. This method can be easily implemented in different experimental animal models and allows a long-term followup of the underlying biological processes and therapies in vivo.

While attempting this procedure, it's important to remember that each optic imaging probe has a different injection procedure and uptake time. Therefore, longitudinal studies should be completed for each new fluorescent dye and optical imaging probe. Once mastered, this technique can be completed in up to five animals in less than 10 minutes if it is performed properly.

This method is able to provide insight into the in vivo mechanisms of inflammation, but it can be also applied to investigate other diseases like cancer or neurodegeneration. After watching this video, you should have a good understanding of how to use in vivo optical imaging with an activatable imaging probe in models of inflammatory diseases.