新生児マウス脊髄圧縮損傷モデル

The overall goal of this procedure is to perform a reproducible spinal cord compression injury in a neonatal mouse. This is achieved by first deeply anesthetizing the mouse. Then performing a dorsal laminectomy to reveal the spinal cord.

And finally by making the compression injury. We have recently established a neonatal mouse model for spinal cord injury and we have demonstrated that neonatal mice exhibit an astonishing degree of adaptive plasticity. Studying this mechanism underlying recovery may provide important insights, which could be used in the treatment of spinal cord injury.

In this video, we will demonstrate how to perform a spinal cord compression injury in a one day old mouse. This surgical procedure, includes anesthesia, laminectomy and two clip driven epidural compressions. Preparing for the surgery.

The surgery is performed under a stereo microscope. Place a warming pad under the microscope. It is crucial to keep the pup warm to avoid sudden death during the surgery.

Place a surgical blanket or a large sheet of clean soft lab paper over the warming pad. For presurgical analgesia, prepare an insulin syringe, 0.3 milliliters 30 gauge, filled with the local anesthetic, Marcaine, diluted with sterile PBS, to a final concentration of 2.5 milligrams per milliliter. For skin incision, muscle removal and laminectomy, use a micro knife, fine forceps and fine iridectomy scissors.

Note that here, we also use forceps that we have bent at the tips to form teeth. These work better than commercially available toothed forceps because the teeth need to be very small. For the spinal cord compression, use a specially modified Yasargil Temporary Aneurysm Miniclip, which we will describe later.

A small length of plastic capillary tubing is also necessary to make a stopper to prevent full closure of the clip, and a clip applicator for placing the clip. For suturing use a needle holder and small scissors to cut the thread. For pain treatment, dilute Temgesic in sterile PBS, to 0.03 milligrams per milliliter.

The spinal cord injury involves a clip driven compression, using a Yasargil Temporary Aneurysm Miniclip. Prior to use, the clip needs to be modified mechanically, by trimming the thickness of the blades down to a 150 micrometers using a sharpening stone. Just before surgery, cut a short stretch of plastic tubing and place it over one of the clip blades.

The tubing acts as a stopper to prevent full closure of the clip. Anesthesia and analgesia. For gas anesthesia, we use a suprane vaporizer connected to an oxygen tank.

Isoflurane is the anesthetic that is vaporized in pure oxygen. To distribute the Isoflurane, Oxygen mixture to the mouse, you need to build a nose mask adapted to neonates. Neonatal mice are so small, that the mask can be made from the tip of a syringe, which serves as the inlet for the plastic tubing connected to the outlet of the vaporizer.

On the side of the mask, drill a small hole to connect an evacuation tube that ends either in a regulated vacuum pump, with a collection unit, or simply in a fume hood. Make an anesthesia overflow chamber using a 150 by 25 milliliter plastic petri dish. On one side, make a hole large enough for the head of the mouse.

On the opposite side, make two smaller holes of the diameter of the plastic tubing. Lastly, make an evacuation hole, smaller than the others, on the top of the dish. Cut the tip of a 250 microliter pipet tip and introduce it into this last hole.

The pipet tip functions as a connector for the evacuation tube that is connected to a vacuum device set for a slight negative pressure. Using Plasticine, fasten the mask just inside the hole for the mouse's head. Using tape or Plasticine, secure the plastic tubing to the ceiling of the chamber, making sure that the tubes are well positioned in the appropriate holes.

Place the chamber on the surgical blanket under the microscope. The entire system is represented in this schematic. A three-way stopcock is placed on the output of the vaporizer.

Here, the path to the sleep chamber is free, while the path to the mask is blocked and the mouse can be placed in the sleep chamber for initiating the sedation. Turning the three-way stopcock to the right allows the gas to flow to the mask and blocks the path to the sleep chamber. In this configuration, the mouse can be transferred to the mask.

Leakage of gas is removed by vacuum from the overflow chamber. Select a mouse pup that has recently been drinking milk, as evidenced by white color visible through the skin. Place the pup into the sleep chamber and introduce vaporized Isoflurane at 4%to achieve a quick sedation.

Transfer the pup to the mask. Carefully position the mouse's snout well into the mask to ensure sufficient exposure to the anesthetic gas. Turn the three-way stopcock, to divert the gas towards the mask.

Inject subcutaneously, 50 microliters of Marcaine at 2.5 milligrams per milliliter, at the desired site of surgery. Be careful not to inject too deep, as blood vessels can easily be hit and introduction of the Marcaine into a vessel is lethal. Wipe off the excess Marcaine with a small sterile compress.

Dorsal laminectomy. Reduce the Isoflurane concentration to one to two percent. After a few minutes, pierce the skin with a micro knife and make a eight to nine millimeter transference opening by gently pulling on the skin in a rostral caudal direction.

A transference opening is much more convenient and gives better lateral access for performing the laminectomy. Retract the edges of the skin incision from underlying tissue, by inserting pieces of spongistatin, subcutaneously rostral and caudal to the incision. Cut the prevertebral muscles with thin scissors to expose the spine.

Note that in the neonate, the spinal process is not fully developed. Identify the midline and cut the lamina transversely with the thin scissors. Pay attention not to cut too deep, as this can potentially damage the dura and even the spinal cord.

Carefully place one blade of a thin forceps between the lamina and the dura. Grab the lamina by closing the forceps and lift it carefully until it detaches, leaving the dura in tact. Repeat the step, two to three times to obtain a one to two segment laminectomy.

Control bleeding using small pieces of spongistatin. Using the thin forceps as rongeurs, remove parts of the facet joints bilaterally to gain enough space to place the modified aneurysm miniclip within the vertebral canal. This step often results in bleeding that can be controled using small pieces of spongistatin.

To improve visibility it is sometimes an advantage to rinse with warm sterile PBS, 37 degrees centigrade. Generating the spinal cord compression injury. Open the clip and place the blades on either side of the spinal cord in the spaces between the facet joints and the cord.

Make sure that the blades are inserted deeply enough to encompass the ventral part of the spinal cord. Rapidly close the clip, while holding the applicator in place, to avoid the clip sliding backwards. After 15 seconds, rapidly release the clip.

Clean the wound using spongistatin. To achieve symmetrical compression, reverse the blades and perform a second compression for 15 seconds in the opposite orientation.Suturing. Remove the pieces of spongistatin that were placed under the skin at the start of the surgery and close the skin with 6.0 sutures.

Inject 0.05 milliliters, Temgesic, at 0.03 milligrams per milliliter. subcutaneously with a 30 gauge insulin syringe and place the pup in a temperature controlled environment at 30 degrees centigrade for a few hours. To reduce aggressive maternal behavior toward the operated pup during the first night, after surgery, prepare a solution of Diazepam, diluted in clean PBS, to 0.1 milligrams per milliliter for intraperitoneal injection at eight grams per kilogram.Results.

As shown by this graph, six hours after spinal cord compression, at lower thoracic levels, the hindlimbs of injured mice are paralyzed, while the forelimbs are not. In the case of sham control animals, that only received a laminectomy, both hindlimbs and forelimbs are highly mobile. The section of the spinal cord shows residual blood at the sight of injury.

In addition, the tissue is translucent, suggesting tissue loss, which often leads to the formation of a cavity several days later.Conclusion. After this video you should have a good understanding of how to generate a spinal cord compression injury in a neonatal mouse. The advantage of using neonatal mice is that we can use specific methods to address adaptive plasticity, which cannot be used in adult mice.

In addition, the model provides a platform for testing therapeutic approaches, such as, stem cell transplantation.