The overall goals of the following experiments are to demonstrate in vivo imaging of collagen remodeling activities, as well as ex vivo visualization of collagen and histological tissue sections by hybridization of collagen mimetic peptides. This is achieved by the folding of a caged collagen mimetic peptide into a triple helix only after UV irradiation to image collagen remodeling in vivo caged collagen mimetic peptides labeled with near infrared Fluor force are activated by UV irradiation immediately prior to intravenous injection, which cleaves the cage group and triggers the CMP to hybridize with collagen strands by forming a triple helix for ex vivo tissue staining, carboxy fluorescein labeled caged collagen mimetic peptides are directly photo activated on fixed tissue sections, which lead to collagen mimetic peptide hybridization with the denatured collagen strands present in the tissue section. Results from near infrared fluorescence imaging and fluorescence microscopy analysis show CMP targeting of denatured collagen strands in remodeling tissues in vivo, as well as in fixed tissue sections to prepare photo activated CMPs for IV injection.
First turn on the UV lamp while the lamp is warming up. Slowly aspirate the freshly prepared near infrared dye, labeled caged CMP peptide solution into a 0.5 milliliter insulin syringe with a transparent barrel and a 28 to 30 gauge needle. Then after carefully removing any air bubbles, place the syringe directly under the UV lamp for five minutes to photo activate the peptide.
Meanwhile, position the mouse in a restrainer under a heated lamp. Label the tail with a permanent marker and then disinfect the tail with 70%ethanol immediately after photo activation. Inject 100 microliters of the peptide solution into the tail vein at the posterior end of the tail.
Place the mouse back into the cage after injection to image collagen remodeling activity in vivo. First, use the pearl Impulse 2.0 software to set the heater plate of the imaging bed of the impulse imager to 37 degrees Celsius. Then open the pearl imager drawer and place an anesthetized nude mouse on the imaging bed.
Once the mouse is in place, close the drawer. Then when the instrument indicates ready on the image software panel, click the 700 channel white light and 85 micron boxes followed by the acquire image button. When the recording has completed, open the drawer, turn the mouse, and acquire images from another angle to acquire high resolution images.
24 to 96 hours after injection. Use surgical forceps and scissors to remove the skin of the mouse and then image the animal by near infrared fluorescence as just demonstrated the acquired near infrared fluorescence. Images can then be analyzed to visualize the collagen and tissue sections.
Place the prefixed room temperature equilibrated mouse cornea slides into a humidified chamber. Then incubate each slide in 0.5 milliliters of blocking solution at room temperature. After half an hour, block the slides on a paper towel to remove the blocking solution and then cover each tissue section with approximately 100 microliters of carboxy fluorescein labeled cage CMP solution for two minutes.
While the CMP solution is permeating the tissue, warm up the UV lamp, then expose the CMP covered tissue sections to the UV light for six minutes to de protect the caged CMPs. After the UV exposure, cover each slide with a piece of param to prevent the tissue sections from drying out and store the slides in the humidified chamber at four degrees Celsius for two hours. After the CMP staining, use a pair of forceps to gently remove the param.
Block the slides on a paper towel to remove any excess CMP solution, and then incubate each tissue section in approximately 100 microliters of freshly diluted dappy solution for one minute at room temperature. Next, immerse the slides three times in PBS in a staining jar for five minutes using fresh PBS for each wash. Then add a drop of mounting medium to each tissue section and place a glass cover slip onto each slide.
Taking care to avoid trapping air bubbles, protect the slides from light in a cardboard slide tray. Finally, image the collagen strands under a fluorescent microscope. In this first set of images, a typical result for the distribution of photo triggered IR dye six 80, labeled caged CMP and a healthy female SKH one nude mouse.
24 to 96 hours after injection is shown. Note the apparent CMP accumulation in the skeleton 24 hours after injection, after which most of the unbound peptides have largely been cleared at 96 hours post injection. The near infrared fluorescence image of a skinned mouse clearly demonstrates the skeletal uptake of IR D six 80, labeled CMP in the spine ribs, as well as within the knees, ankles, wrists, and lower mandibles.
In ex vivo tissue staining, photo triggered fluorescently labeled caged CMPs, specifically hybridized to denatured collagen strands in tissue sections. In these final images, CMP staining clearly reveals the fine parallel collagen fibrils of the corneal stroma validating the use of CMP as a collagen specific staining agent. The demonstrated imaging methods based on CMP collagen hybridization provide researchers with a new tool for studying the tissue remodeling process and could lead to development of new diagnostics for diseases associated with high collagen remodeling activity such as cancer, osteoporosis, arthritis, and fibrosis.
