The overall goal of this in vivo duo-color two-photon imaging method is to monitor the acute spinal vascular dynamic changes that occur following a contusive spinal cord injury. This method can help answer key questions about how these dynamic vascular changes impact the pathological progression and the functional deficits that occur after spinal cord injury. The main advantage of this technique over traditional techniques using promodern samples is that it provides evidence of the acute vascular dynamics of spinal cord injury within a living animal.

The implications of this technique extend toward the treatment of spinal cord injury, as this method allows us to examine potential neuroprotective strategies aimed at early vascular repair. We first had the idea for this duo-color imaging method when we found that sequential injection of contrasting fluorescence colors allow us to visualize disrupted vessels after spinal cord injury. Before beginning the procedure, confirm the appropriate level of sedation by lack of response to toe pinch of a six-week-old female Sprague Dawley rat followed by subcutaneous injection of analgesia and an anti-inflammatory agent.

Shave the rat in the cervical spine region and in the neck region on the breast side of the animal, and swab the exposed skin with a Betadine surgical scrub and 70%alcohol wipes. Apply ointment to the animal's eyes, and place the rat in the supine position on a sterile surgical pad on top of a 37 degree Celsius heating pad under a sterile microscope. Find the pulse point near the collarbone to locate the external jugular vein, and use a pair of small sterile spring scissors to make a vertical incision at the intersection of the caudal ramus of the right mandible, the greater tubercle of the humerus, and the manubrium.

Use the spring scissors and fine forceps to isolate the jugular vessel, and use a sterile surgical suture line to ligate the distal end of the vessel. Next, use a pair of micro scissors to make a small incision in the vessel wall, and slide a specialized catheter made from a 21 gauge needle attached to a one milliliter syringe filled with saline into the vessel. If a small amount of blood enters the needle, the insertion was successful.

Secure the needle at its proximal and distal ends. To stabilize the spine, switch the animal to the prone position, and use a number 15 scalpel blade to cut the skin along the midline at the desired spinal levels. Locate the second thoracic vertebra by the spike between the scapulae and count upward from T2 to find the C7 vertebra.

Dissect the muscle layers from the fifth to the seventh cervical vertebrae bilaterally to expose the lateral facets, and make a slit on both sides of the lateral vertebral bone. Slide the stainless steel arms of a modified stabilizing apparatus under the exposed transverse process facets, and tighten the screws to stabilize the spine. Then carefully remove the C5 to C7 laminae and place a small piece of saline-soaked gel foam on top of the exposed dura mater to keep it hydrated.

To create the two-photon imaging window, place small pieces of gel foam into the gap between the muscles and the vertebral bones and a thin line of gel foam between the spinal cord and vertebral bones. Then seal the surrounding muscle bone area with tissue adhesive glue. the most critical step in preparing a successful imaging window is making sure that there isn't any minor bleeding inside the window.

After allowing the glue to dry for about five minutes, fill a sterile one milliliter syringe with warm 4%agar, and dispense a wall of agar along the edge of the window. The agar will solidify quickly while remaining flexible. To obtain a baseline image, first remove the gel foam and add immersion fluid inside the window.

Place the stabilized animal into the two-photon microscope dark chamber with the two-photon imaging window directly under a low magnification objective, and lower the lens carefully into the window. Fill a one milliliter sterile syringe with 0.5 milliliters of freshly prepared Rhodamine B isothiocyanate dextran solution, and connect the syringe to the catheter. Depress the plunger very slowly to deliver the dye using the eyepiece to identify the area of interest, and use a charge-coupled device camera to acquire a landmark bright field image of the surface blood vessel patterns.

Next, open the two-photon imaging software to turn on power and select appropriate laser excitation wavelength. Then select the proper two-photon laser power, HV level, and fluorescent channel that corresponds with the injected fluorophore experiment and image the tissue through the imaging window to collect both images and line scan data. To introduce the C7 midline contusion injury, place the rat on a Louisville Injury System Apparatus device, and select a 0 point setting.

Then adjust the tissue displacement, and click run experiment to trigger the impact or release creating the injury. Next place a small piece of saline-soaked gel foam on top of the exposed dura mater to keep it hydrated and open new images through the imaging window using the same imaging parameters. Transfer the animal back to the dark chamber, and inject 0.5 milliliters of freshly prepared fluorescein isothiocyanate dextran into the catheter.

Then return the animal to the microscope dark chamber and obtain images in both fluorophore channels. Rhodamine dextran fluorescent dye injection allows the capture of a landmark image for mapping of the vascular network at baseline. After the generation of a moderately severe C7 midline contusive injury, the introduction of FITC-dextran facilitates the detection of the vascular structure after injury-induced leakage of the first dye into the surrounding parenchyma.

Imaging of the vessel before and after injury also allows changes in the vessel diameter and response to injury to be quantified as well as changes in the pre-and post-injury red blood cell velocity through the vessels to be calculated. Taken together, these data can be used to classify the imaged vessels as veins or arteries. While attempting this procedure, it's important to remember to avoid noticeable breathing artifacts, and to try to limit any minor bleeding within the image window.

This procedure can also be performed on transgenic animals to answer additional questions about the neurovascular interactions that occur following spinal cord injury. After watching this video, you should have a good understanding of how to use two-photon microscopy to monitor acute vascular dynamic changes after a contusive spinal cord injury in a living animal.