Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Transcript

The overall goal of this protocol is to use an advanced dorsal skinfold chamber to examine the development of tumor-associated vasculature in detail in a mouse model. This method can help to answer key questions in the cancer field, such as improvement of therapy and understanding tumor progression. The main advantage of this technique is that processes are studied in the right context, in true life and that these processes can be studied over time.

This procedure will be demonstrated by Doctor Ann Seynhaeve, the senior researcher in my lab. From a non-transgenic donor mouse, harvest a 10 mm diameter non-necrotic tumor fragment for implantation into the dorsal window chamber of the recipient mouse. Use a normal mouse donor for a syngeneic tumor or immunodeficient mouse donor for a xenograft.

The recipient mouse must be at least 12 weeks old and weigh twenty or more grams. Perform all the surgical procedures under aseptic conditions. For example, autoclave the surgical instruments and chamber materials.

After sedating the recipient mouse, remove the hair from the entire dorsal side using an electric trimmer then use hair removal gel. Rinse the skin clean and dry it. Finally, scrub the depilated skin clean with ethanol.

Now, mark a horizontal center-line along the spine. Then, position the view window at least 2 mm below the line and mark the window's perimeter on the skin. Next, using a scalpel and scissors, carefully remove the skin within the perimeter line without damaging the underlying fascia.

Then, pull up the skinfold and use an ear puncher to introduce 1 mm holes on either side of the window area for the bolts that hold window in place. Position the back frame on the mouse and secure the frame by placing the nuts on the bolts. Next, pull the skin two to three millimeters over the frame to make sure it fits.

If so, poke 23 gauge needles through the two suture holes at the top of the frame. Secure the frame by tightening the nuts and hold them with clamping needle holders do not over tighten the nuts and prevent the skin from turning around the bolts. Next, replace the needles with sutures to secure the frame to the skin.

Now turn the mouse to the side. From this angle introduce a thick piece of filler glass and a standard 12 millimeter cover glass. Secure both with a retaining ring so that the fascia tight against the glass.

The next step is to hydrate the window using 0.9%sodium chloride. Allow a few drops to fall onto the view area and soak up the excess. Now, just below the window, use a 25 gauge needle with a 90 degree bend to make a deep pocket in the fascia that can hold a cubic millimeter of tumor tissue.

Then insert the tumor tissue into the pocket. Finally, close the window view area using a standard 12 millimeter cover glass and a retaining ring. Any air bubbles will dissipate within hour.

Prior to sedating the mouse, warm a custom-made temperature controlled platform to 37 degrees Celsius. After sedating the mouse, position it on the warmed platform. Attach a nosecone to deliver isoflurane and if the evaluation will take more than five minutes, apply a ophthalmic appointment.

Now, bolt the chamber using the window's bolts to custom-made holder and secure the holder to the platform. Then, clean the glass window with water and cotton. Now, at the microscope, check for vessel development in the tumor using the 10 times objective, under bright-field and fluorescent light.

After the gross examination, turn on the confocal lasers. Set the microscope control panel to a smart gain of 100 volts per turn. A smart offset of 10%per turn, medium zoom medium X and Y positioning and 100 microns of Z position per turn.

Set the acquisition mode to XYZ or XYZT. Select a resolution. Choose an acquisition speed of two or 400 Hertz.

Set the pinhole to one airy unit. And select the bidirectional X option. When evaluating multiple fluorophores with a possible overlapping spectral range, select sequential scan inbetween frames to prevent bleedthrough and set the correct beam paths.

Set the line average to four and lastly, select a new data set for the experiment. Now, focus in on the tissue of interest in doing so, it is best to use dry lenses with higher numerical apertures. Next, use the fast live scan to get the best possible signal to noise ratio by adjusting the gain and offset to where there is no overexposure.

Now, select the Z stack in the acquire tab. Make a fast Z scan. From the fast scan, decide on the beginning and end positions of the full scan.

Then, set the Z step size according to the pinhole size and proceed with the full scan. Host mice expressing fluorescent labels were imaged after tumor implantation using the described protocol. The eNOStag-GFP label expresses in the Golgi and the cell membrane of endothelial cells.

The ROSAmT/mG label expresses red florescence ubiquitously and green florescence in Cre Recombinase expressing cells. One application of this technology is for the study of vessel development as all stages are seen in tumors. For example, endothelial fronts can be seen sprouting into unvascularized areas.

Tumors also have damaged vessels and established vascular beds with mature branched vessels. Another application is to study lumen formation, bloodflow and extravasation by applying labels to the blood, such as nanoparticles that fluoresce. Thus, blood flow can be observed in the lumen of vascular tubes and close to endothelial tip cells.

In the tip cells, formation of a lumen the stock is observable. Intra-vital microscopy can also be used to investigate the effect of chemotherapeutic agents. For example, 24 hours after nanoparticle injection into an eNOStag-GFP animal, the nanoparticles remained in the vasculature with minimal extravasation into the tumor interstitium.

However, when nanoparticles were administered in combination with TNF, extravasation was observed into the tumor interstitium. This is evidence for an increase in inter-tumoral drug delivery. After watching this video, you should have a good understanding of how to install a dorsal skinfold window chamber and how to study tumor-related processes using confocal microscopy.

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Journal (Medicine)

Journal (Medicine)

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

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