Simple and Effective Procedure for Hemostasis in Mouse Arteries

This research develops a method to seal arterial punctures while preserving blood flow. It investigates whether fat wrapping prevents ischemia after arterial puncture. A key experimental challenge in this procedure is achieving effective hemostasis after arterial puncture without causing arterial occlusion.

To begin, position the anesthetized mouse on the surgical table. After confirming anesthesia, remove hair from the cervical and cranial regions using a depilator. Disinfect the exposed skin with povidone iodine, followed by 75%ethanol to eliminate any residue.

Place the mouse on a heating pad set to 37 degrees Celsius and keep it there for the entire duration of the experimental procedures. Reposition the mouse in a supine position on the heating pad. After making a midline cervical incision, excise a three-by-three-by-one-millimeter subcutaneous adipose tissue graft for future use.

Perform blunt dissection of the submandibular gland, sternal hyoid muscle and sternocleidomastoid muscle to expose both common carotid arteries. Then carefully free the common carotid artery to expose the internal and external carotid arteries. Place vascular clamp one on the distal end of the common carotid artery, and then place vascular clamp two on the proximal end of the artery.

Next, puncture the common carotid artery between the two clamps using a 23-gauge needle. After removing the needle, wrap the common carotid artery at the puncture site using the cut surface or non-serosa side of the previously harvested fat pad. Place a 4-0 suture beneath the fat-wrapped artery.

Gently tie a single throw over the fat surface to apply pressure for secure contact between the fat pad and the vessel wall. Then adjust the ligature gradually to balance pressure and achieve hemostasis. Elevate the distal common carotid artery using a moistened cotton swab.

Release vascular clamp one, and slowly withdraw the swab to allow a gradual return of blood flow toward the puncture site. Now, check for blood flow at the puncture site. If active bleeding is observed, reapply vascular clamp one on the distal common carotid artery.

After one minute, slowly release vascular clamp one. If bleeding persists, reposition the fat pad or slightly tighten the ligature to increase pressure until full hemostasis is achieved. Elevate the proximal common carotid artery using a moistened cotton swab.

Next, release vascular clamp two and slowly withdraw the swab to permit controlled blood flow toward the puncture site. If bleeding occurs, reapply vascular clamp two on the distal common carotid artery. Now, slightly tighten the single throw of the suture to increase pressure and wait for one minute.

Then elevate the proximal common carotid artery and release vascular clamp two by slowly rolling the moisten swab toward the puncture site. Add another throw using the same suture to form a double knot over the initial throw, improving fat pad fixation and promoting balanced hemostasis. Confirm that the common carotid arteries on both sides of the fat pad are filled and maintain consistent vessel diameter, indicating successful blood flow restoration.

Finally, suture the cervical incision and apply an antiseptic to the wound site. Pre-and postoperative laser speckle contrast imaging and perfusion unit measurements in both sham and operated mice showed no significant changes, confirming that cerebral blood flow was not compromised by the arterial hemostasis technique. Histological examination of brain tissue by hematoxylin and eosin staining at seven, 14 and 28 days post-operation in operated mice and in sham mice showed intact tissue architecture in the cortex, hippocampus and thalamus without pathological changes.

Nissl staining showed organized neuronal alignment, normal cellular structure and preserved nissl bodies across all groups and time points, indicating no structural damage due to arterial closure. The present method aims to seal the puncture site post-procedure, while maintaining normal blood flow, thereby preventing postoperative ischemia.