Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

The overall goal of this procedure is to develop an easy and highly reproducible model of spinal cord ischemia in mice. This is accomplished by first surgically dissecting the dorsal surface of the mouse to expose the T nine through T 12 vertebrae. In the second step, a high speed drill is used to thin the dorsal surface of the T 10 or T 11 vertebrae exposing the posterior spinal vessels.

A photo active dye is then administered via the retroorbital root to label the blood vessels. In the final step, the posterior spinal vessels on the dorsal surface of the spinal cord are irradiated to induce photo thrombosis in the exposed blood vessel. Ultimately, the outcome of the spinal cord injury is assessed by nissle, staining and immunohistochemistry.

The implications of this technique extend toward therapy of spinal cord ischemia as it can be used to test the efficacy of various neuroprotective drugs aimed at improving cell survival and recovery after spinal cord injury. Demonstrating the procedure will be Nana, a research scholar myself, ri both from dr. She's lab.

Before beginning the surgery, prewarm the metal allied lamp for 30 minutes to stabilize the lamp power. Then using the 10 x objective and theoretical, adjust the field diaphragm in an upright FN one epi fluorescence microscope to set the size of the illuminated region to 0.75 millimeter diameter. Next, apply artificial tear ointment to both eyes of an anesthetized mouse and place the animal onto a prewarm heating pad.

Confirm the appropriate state of anesthesia by a lack of response to toe pinch. Then use an electric hair trimmer to clip the hair on the animal's dorsal surface around the midline. Scrub the surgical site with 70%ethanol, followed by Betadine solution three times, and then drape the animal.

Now, place the animal in the prone position on a homeo themic heating pad on a surgical platform using a snout clamp to secure the animal and to maintain an elongated neck region. Then begin the surgery by making an incision along the dorsal midline from the T nine to the T 12 thoracic vertebrae. Moving aside the skin to expose the surgical area using a scalpel, carefully clear the muscle to expose the dorsal spines of the T nine through T 12 vertebrae, stopping the bleeding as necessary with the gentle application of a sterile cotton swab.

Next, separate the T 10 through T 12 vertebrae from the surrounding muscle and secure them with a vertebral clamp to stabilize the animal and to prevent any movement using a high speed drill equipped with a bone polishing drill bit. Now, carefully and gently thin the dorsal surface of the T 10 or T 11 vertebra to visualize the posterior spinal vein and other small vessels on the dorsal surface of the spinal cord. Take care during this step as excess pressure might cause the drill to enter into the spinal cavity and damage spinal cord, while uneven thinning of the bone may result in improper elimination and production of irregular infarc.

To prevent thermal damage during the thinning procedure, apply a gentle and constant stream of normal saline along with constant suction to remove the debris. Then using a scalpel, carefully smooth the bone surface until the main vessel is clearly visible. Taking care not to damage the spinal cord.

Once the blood vessel is visible, use an insulin syringe to administer the photo active dye through the retroorbital sinus. Three minutes after the injection, measure the blood flow with a laser doppler flow meter to induce photo thrombosis. Place the animal on an XY position, adjustable stage over a height adjustable lab jack moving the mouse until the exposed region of the spinal cord is directly under the 10 x objective of the microscope.

Set the power of the light source to 12%and irradiate the exposed region in the middle of the thinned spinal cord through the 10 x objective for two minutes. Obtain images at the beginning and the end of irradiation recording the time of each image capture. If no hemorrhaging is observed, use an absorbable four oh size suture to close the superficial fascia along with the muscles on either side of the spinal cord, followed by closure of the skin with the same, then apply antiseptic to the edges of the skin and place the animal back onto the heating pad.

When the animal has fully recovered. Check for signs of neurological deficits by observing the movement of both hind limbs to confirm the induction of ischemia. Changes in the blood flow can be measured with a laser doppler flow meter before and after photo thrombosis.

As just demonstrated in this experiment, the blood flow dropped to about 20%immediately after the light illumination compared to the basal level here, fluorescent images of the spinal cord blood vessels at the beginning and end of the photo thrombosis are shown. Note the formation of a blood clot after the two minute photo period suggesting the induction of ischemia and consistent with the reduced blood flow measured by the laser doppler flow meter. In this series of rostral toco spinal cord transverse sections of a normal and photo thrombosis induced epicenters three days after photo thrombosis, a clearly demarcated infarct region indicating the presence of spinal cord tissue damage and cell death in the photo illuminated tissues can be observed.

Indeed, immuno staining of the spinal cord tissue three days after thrombosis revealed a loss of new end positive neurons in the gray matter of the ischemic core. While the GFAP expression was increased in the ischemic core border, the IBA one positive microglia also exhibited a G OID morphology, as well as an increased IBA one expression with an overall increase in the GFAP and IBA one expression in the entire per infarct region indicating neuronal death and reactive gliosis in the penumbra after spinal cord ischemia. Further substantial functional deficits such as disabled hind limb movement one day after photo thrombosis were also observed in the injured animals.

Following this procedure, a methods like two photo microscopic can be performed to answer additional questions like, what are the roles of axonal degeneration, regeneration, and neuronal acetic calcium signaling in spinal cord ischemia? After watching this video, you should have a good understanding of how to thin the dorsal surface of T 10 RT 11 vertebrae, and how to eradiate the exposed posterior spinal blood vessels.