To begin, arrange all the materials required for mouse surgery on the surgical table. Place the anesthetized mouse on the table, ensuring the pillow is under its neck. Disinfect the surgical site by alternating circular swabs of betadine or chlorhexidine and alcohol three times per disinfectant.
Perform a toe pinch to confirm the depth of the anesthesia. Using surgical tape, restrain the fore limbs of the mouse. Under a dissecting microscope, bring the ventral surface of the mouse's neck into view and adjust the magnification to observe the surgical site comfortably.
After making a one-centimeter longitudinal midline incision, insert the tip of the closed scissors into the incision and gently open it to perform a blunt dissection of the subcutaneous connective tissue, separating the two salivary glands. With the help of fine forceps, gently pull the right salivary gland through the incision to rest on the exterior surface, or use a blunt hook retractor to retract the salivary gland laterally. Continue with the blunt dissection of the connective tissue until the sternocleidomastoid and the digastric muscles are visible.
Using angled-tip forceps, continue the careful dissection of connective tissue near the caudal end of the muscular triangle to expose the jugular vein, vagus nerve, and common carotid artery, or CCA. Continue dissecting the connective tissue around the CCA from the base of the muscular triangle to the omohyoid muscle. Use small cotton balls to control any minor bleeding and to absorb secreted fluids from the salivary glands.
Dissect connective tissue to separate the CCA from the vagus nerve, taking special care to minimize handling of and damage to the vagus nerve. After mobilizing the CCA, place a one-centimeter piece of 6-0 suture on the sternum and pass the angled-tip forceps under the CCA. With the angled-tip forceps in the right hand and fine forceps in the left hand, pass the suture to the angled-tip forceps and grasp near the end of the suture.
Gently pull half of the length of the suture under the CCA. After placing a second suture, loosely tie each suture around the CCA without tightening the knots or restricting blood flow. Using the angled-tip forceps, carefully remove connective tissue at the top end of the muscular triangle above the omohyoid muscle to locate the CCA and its bifurcation into the external carotid artery and internal carotid artery.
Then place a piece of suture on the sternum and carefully clear away connective tissue from all sides of the external carotid artery near the bifurcation. Pass the angled-tip forceps under the external carotid artery. With the fine forceps in the left hand, pass the suture to the angled-tip forceps in the space between the internal and external carotid artery and gently pull half of the suture's length.
Loosely tie the suture around the external carotid artery without tightening the knot. To prepare the bent needle, hold a 33-gauge half inch needle with the bevel facing up. Grasp the tip of the needle with a sterile needle driver and bend it approximately 30 to 40 degrees towards the bevel.
Tighten the knot of the suture around the external carotid artery. Then slide the lower suture on the CCA towards the sternocleidomastoid muscle as far as possible, and tighten the knot. Place a cotton ball at the edge of the cavity to absorb secreted fluid and blood during the injection.
Holding the syringe in the right hand and the fine forceps in the left hand, bring the needle to the artery immediately above the lower suture on the CCA. With the fine forceps, gently pull the loose end of the lower suture in a caudal direction to place a low level of tension on the CCA. Insert the needle into the CCA just past the bevel and slowly release tension from the suture.
With the left hand, push the syringe plunger to slowly inject the solution containing the agent of interest or control. Then grasp the loosely-tied upper suture on the CCA with the fine forceps and lift to kink the artery. Remove the needle and set the syringe aside.
With the angled-tip forceps in the right hand, keep the kink in the artery and tighten the knot in the upper suture on the CCA. Using cotton balls, absorb any residual blood within the surgical cavity. Locate the injection site on the CCA and determine the number of sutures needed to close.
Irrigate the injection site and lumen of the isolated area of the CCA thoroughly with sterile saline to remove coagulated blood. Using the angled-tip forceps, grasp the 9-0 suture needle close to the sewage. Hold the artery and gently press the CCA sides together with the fine forceps.
Then pass the needle and suture through both sides of the artery with a single bite. To close the incision site with a surgeon's knot, perform an instrument tie with the fine forceps and angled-tip forceps using a minimum of four throws. Using the fine forceps, untie and remove the suture around the external carotid artery, followed by an upper suture on the CCA to restore circulation.
Slowly loosen the lower suture on the CCA, but do not immediately untie it. After confirming the injection site is sufficiently closed, remove the upper and lower CCA sutures. Reposition the salivary gland in the cavity and, using a sterile suture pack, close the incision with one suture placed every three to four millimeters.
After intracarotid injection of GFP-labeled bone marrow human mesenchymal stem cells, immunohistochemistry of brain sections revealed the presence of GFP-positive cells, indicating the homing of bone marrow human mesenchymal stem cells to the gliomas. No significant difference in the GFP human mesenchymal stem cell homing efficiency between the CCA ligation and CCA repair procedures was observed. The dispersion of GFP human mesenchymal stem cells within the tumor revealed no significant difference between the median counts of GFP positive cells in high-powered fields between the CCA ligation and CCA repair groups.
