Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

The overall goal of this procedure is to observe movement and interaction between microglia, monocytes and other cell types in the injured spinal cord. This is accomplished by first creating bone marrow chimeric mice that will have either resident microglia or bone marrow derived macrophages labeled with a fluorescent marker. The second step is to produce a dorsal column crush injury in the spinal cord.

Then the animals are reopened and the spinal cord is stabilized For multi photon imaging, the final step is to take either single images or time-lapse movies within the lesion. Ultimately, image analysis shows cellular movement and cellular interaction with surrounding substrates. The main advantages of this technique over traditional technique, such as cell culture and fixed tissue, is that cells can be imaged in their natural complex environment in the living animal.

For this surgery, use chimeric or double transgenic animals. See the text protocol on how to label resident or bone marrow derived cells after inducing anesthesia, maintain it with one to 2%ISO fluorine for a breathing rate of about 60 to 100 breaths per minute. Now, apply eye ointments and confirm a surgical plane of anesthesia with a toe pinch.

Prepare for the surgery by first shaving the targeted spinal cord area, and then wiping the skin clean with povidone iodine and 70%alcohol scrubs, drape the animal and make a hole to access the surgical site. Then lay out the autoclave tools on a sterile drape. This includes a number four forceps that is fixed to be one millimeter between the tines and has marks one millimeter from the tips.

Now could open the skin at the midline using iris scissors, exposing the musculature. Proceed with the surgery under a dissecting microscope. Along the midline, cut the back muscles where they attach to the dorsal spinal processes with the goal of exposing the spinal column.

This involves cutting the trapezius and ligaments of the latisimus dorsi. Next, remove the rotator muscles between the transverse processes and the dorsal process to reveal the lamina of the specific vertebra targeted for removal. Here T 10 is exposed.

Proceed by inserting the RO juror tips into the space above and below the process. Then lift slightly to make sure the tips are securely in place and not too deep. Then close the RO jurors to remove the lamina.

Now, remove all the smaller parts of the remaining vertebra, thus enlarging the opening. Using a 30 gauge needle, poke two holes, one millimeter apart in the dura to make an access point for the fixed forceps. Then insert the fixed forceps at a 90 degree angle through the dura, keeping the marks on the tines above the dura.

Now, pinch close the forceps for 10 seconds. Do the ten second squeeze two more times and remove the forceps. Next, soak a piece of absorbable gelatin, compressed sponge in saline, and cover the injury site with it.

Close the muscle over the gelatin sponge using number four, synthetic non nylon running sutures. Then use five millimeter wound clips to secure the skin. Complete the procedure with a subcutaneous injection of 10 microliters of bupivacaine in 490 microliters of saline for one milligram per kilogram.

Then inject five microliters of buprenorphine into the gluteus muscle for 100 micrograms per kilogram.Postoperatively. Warm the animal and monitor it until it regains consciousness. To reopen the animal for imaging, induce anesthesia as before, and then remove the wound clips according to the manufacturer's and instructions.

If necessary, apply a hair removal product to the area around the incision. This time, apply extra scrubs with povidone iodine and 70%alcohol. Now, reopen the skin along the previous incision.

Remove the remaining sutures and open the muscles with forceps using scissors as needed. Under the dissecting microscope, use the scissors to make pockets for the spinal cord clamps along the transverse processes on the vertebra, one level above the laminectomy and one level below the laminectomy. Insert the clamps of the spinal cord stabilizer around each vertebra and tighten the clamps.

Adjust the clamps so the spinal cord is parallel with the imaging platform and the tissue is stable. If a breathing artifact is seen, adjust the clamps to minimize the movement. Then cover the clamps and paraform.

Next delicately, remove the gel foam using forceps. Remove as much as possible. Also, remove any thick scar tissue over the meninges that may be present.

Once all the gel foam is removed, cover the spinal cord with saline. If bleeding is noted, use a gelatin sponge to staunch the bleeding. If necessary, use cautery during cautery.

Be careful not to touch the spinal cord and keep it covered with saline and gel foam. Now create a well to hold the immersion fluid, seal the skin and tissues with cyanoacrylate adhesive. Then build up a well of dental acrylic between the two clamps.

Allow the well time to cure. Once cured, fill the well with artificial CSF. Keep monitoring for bleeding during these steps.

At this point, optionally inject fluorescent vessel dies into the tail vein. To highlight blood vessels during imaging, use a fluorescent DExT strand above 70 kilodalton quantum dots or fluorescently labeled lectins. During imaging, maintain the mouse at 37 degrees Celsius in a controlled environment On the microscope stage, The most critical steps to pertaining a successful preparation are making sure that the spinal cord is clean, not bleeding, and that there's no visible breathing artifact.

Periodically during imaging, check the level of anesthesia by monitoring the animal's breathing, which should be between 60 and 100 breaths per minute. Correct the anesthesia by adjusting the ISO fluorine levels. After imaging, return the animal to the surgical site there, remove the spinal clamps and pull the acrylic off the skin.

The mouse can then be closed for later imaging as at the end of the initial surgery or be euthanized five days after injury. The forcep insertion sites are still visible, but the lesion has begun to increase in size due to secondary injury caused by inflammation. The axons at the co end of the lesion have retracted from the initial site of the injury.

Concurrently, a large influx of CX three C one positive cells, either microglia or macrophages can be seen infiltrating in and around the crush lesion to discern the behavior of these cells within the lesion microenvironment, a radiation chimera model was used first. The bone marrow progenitor cells of a CX three CR one positive GFP mouse were replaced with non fluorescent donor cells, so only GFP positive CNS resident microglia would be visible. Secondly, bone marrow progenitor cells in a non fluorescent mouse were replaced by marrow cells from a CX three CR one positive GFP mouse to view bone marrow derived cells.

Before the injury, the only bone marrow derived GFP positive cells detected were largely paravascular, which have been reported to be continually replaced from the bone marrow after the crush injury. CX three C one positive GFP cells are increased along the inside of blood vessels surrounding the lesion. Additionally, the lesion center was filled with infiltrating CX three CR one positive GFP macrophages.

Using this technique, different transgenic animals can be used in order to label other fluorescent cell populations to identify their interactions both within the spinal cord and in other disease models.