The endovascular perforation model combined with postoperative MRI allows verification of the bleeding and exclusion of other intercranial pathologies. One of the advantages of this approach is the option to quantify the bleeding volume for further subgroup analysis. Begin by shaving the neck hair of the mouse.
Apply ophthalmic ointment during the procedure. Sanitize the skin with 70%ethanol followed by Betadine or chlorhexidine and apply 1%lidocaine. Place the mouse in a supine position with its head and torso facing an upward direction.
Stabilize the mouse dorsally by using tape on the four extremities. Stretch the skin of the neck and elevate it. Unfold the neck skin from the chin to the upper edge of the breast bone and separate salivary glands from the adjacent connective tissue.
Expose the common carotid artery sheath by separating the muscles along one side of the trachea. Expose the surgical field with a retractor or sterile gauze swab and detach the carotid artery and leave a free 8-0 silk suture without ligating it, ensuring not to damage the vagal nerve. Dissect the distal end of the ECA and ligate the vessels twice in a distal position.
At the midpoint of the twice ligated filament segment disjoin the ECA and create a vessel stump. Leave a silk suture around the ICA without closing it. Prearrange one ligation for the filament around the ECA stump.
Do not close it until successful filament insertion. Close the ICA and CCA temporarily by using a suture or micro clip. Using microvascular scissors, make an incision in the ECA and insert a 5-0 alternatively 4-O Prolene filament, and advance it into CCA.
Close the ligature on the ECA while detaching the micro clip on the ICA and CCA. Pull back the filament gently and adjust the ECA stump in the cranial direction while invaginating the filament through the bifurcation into the ICA. Place the filament tip medially at an angle of approximately 30 degrees to the tracheal midline in horizontal plane and push it inside ICA.
At the ACA-MCA bifurcation, resistance is observed. Move the filament forward up to three millimeters and perforate the right ACA. Quickly withdraw the filament to the ECA stump allowing blood flow in the subarachnoid space.
Place the filament in this position for 10 seconds. Withdraw the filament. The CCA can be temporarily closed beforehand to avoid excessive blood loss and then withdraw the filament.
Ligate the ECA using prearranged sutures and reopen the CCA if you have closed it before. The presence of muscle tremors, ipsilateral myosis, gasping for breath, altered heart rhythm and urinary incontinence can be supporting evidence of successful surgery. Ensure that there is no bleeding leakage and disinfect the skin.
Suture the wound. Place the mouse in a thermal box and wait until the animal has sufficient consciousness to have sternal recumbency. To relieve the pain, administer 1.2 milligram per milliliter of paracetamol.
Using a rodent scanner on a mouse head resonator perform MRI after 24 hours of surgery, Place the mouse on a heated circulating water blanket at 37 degrees Celsius. First, perform a quick reference scan acquiring three orthogonal slice packages. Then use a high resolution T2 weighted 2D turbo spin echo sequence for imaging.
Transfer the data into the DICOM image format and use Imagej software for SAH grading and volumetry of blood clots. To ensure the correct bleeding site and exclude other intracranial pathologies, MRI scans were performed 24 hours postoperatively. The SAH bleeding grade was quantified based on T2 weighted MRI scans.
Amongst the examined mice, 14%of the animals were classified as bleeding grade zero without radiological evidence for SAH or hemorrhage. The majority of the animals showed bleeding grades between one to three, and 10%showed bleeding grade fourth which was defined SAH along the stroke and/or ICH. The total bleeding volume of each SAH grade, based on SAH thickness, was quantified which helps to determine the bleeding area.
The calculated bleeding volume based on the axial SAH thickness showed significant differences in their corresponding subgroups grade one to three. Throughout the whole surgery, a thorough dissection of surrounding connecting tissue is crucial for correct positioning of the sutures. Make sure that the length of the ECA stump is sufficient so that you've comfortable working space for the ligation, incision and insertion of the filament.
Never push the filament forward when you feel resistance before the MCA-ACA bifurcation to prevent premature vessel perforation.
