The overall goal of this procedure is to induce a standardized subarachnoid hemorrhage in mice. This is accomplished by first connecting the anesthetized animal to monitoring devices to supervise physiologic conditions during surgery. The second step is to apply sensors for cerebral perfusion and intracranial pressure measurement as key parameters for bleeding induction.
Next, the subarachnoid hemorrhage is induced by endovascular filament perforation. The final step is to perfuse the animal and isolate the brain to visualize the extravasated blood distribution. The main advantage of performing this technique in mice instead of Rex is that we can make use of transgenic animals.
Generally, individuals new to this method will struggle because intubation, multimodal physiology monitoring, as well as vessel preparation are quite challenging to perform in mice. Visual demonstration of this method is critical as the surgical steps are difficult to learn. Demonstrating the procedure will be Catherine Shula, a PhD student from my laboratory.
She has more than two years experience in performing this SAH model. To begin place, an anesthetized animal on a slanted platform working under a microscope, retracts the tongue with bent forceps to locate the vocal cords. Once visualized intubate during inspiration with a tube made from a 20 gauge venous catheter.
Next, place the mouse in a prone position and check for the correct placement of the tube with a piece of cotton or a micro capnograph if correctly placed. Connect the intubation tube to the respirator. Ventilate the mouse with room air, supplemented with 25%oxygen.
Connect the intubation tube to the micro capnograph. Maintain the end expiratory partial pressure of carbon dioxide at 30 millimeters Mercury by adjusting the ventilation frequency. After inserting a rectal temperature probe, place the animal on a heating pad to maintain the body temperature At 37 degrees Celsius, place an annular pulse oximeter sensor on the right hind paw.
Next, make an incision from eye to ear. Retract the skin and dissect the left temporal muscle from the temporal bone. Once exposed, glue a laser doppler flow meter Probe onto the left temporal bone.
Hold the probe in a fixed position until the glue hardens. Drill a hole of approximately 1.5 millimeters in diameter into the left temporal bone while drilling. Cool the bone with saline.
To prevent heat damage, insert the intracranial pressure probe into the cranial cavity and push it forward as dorsally as possible to prevent tissue damage and bleeding. Once the probe is placed correctly, fix and seal with cement and let it dry for five minutes. When the cement is dry, turn the mouse carefully to a supine position.
Using standard techniques, catheterize the left femoral artery for continuous blood pressure monitoring. Once in place, connect the femoral catheter to the blood pressure monitoring device. First, open the skin with the pair of scissors from the sternum to chin blunt.
Dissect through the connective tissue and push the salivary glands aside to expose the left common carotid artery. Isolate the CCA from the surrounding tissue, taking care not to damage the vagal nerve located in the same connective tissue sheath. Next, carefully expose and isolate the internal carotid artery and the external carotid artery.
Once isolated, ligate the ECA as far cran as possible, prearranged two additional ligations around the ECA for later use using an applicator occlude, the CCA and the ICA temporarily with micro clips. Gently pull the clips back to make sure that they're applied correctly. For bleeding induction, we use a 12 millimeter long proline five zero filament.
Cut a small hole into the ECA using vessel scissors and insert the filament. Once the filament is in place, close the insertion site using one of the prearranged ligations. Next, remove the micro clips with the micro clip applicator to induce bleeding.
Advance the filament into the ICA until the ICP rises. A sudden rise of the ICP indicates bleeding induction, withdraw the filament immediately and ligate the ECA by closing both prearranged ligations consecutively. This prevents bleeding out of the insertion site.
Suture the skin wound and monitor the physiologic parameters of the animal for another 20 minutes. At the end of this period, remove both the ICP and LDF probes after trans cardial perfusion. Remove the brain and evaluate the blood distribution in the subarachnoid space before bleeding, the ICP is around four millimeters.
Mercury bleeding results in a sharp increase of the ICP up to 120 millimeters. Mercury ICP values then stabilize within five minutes at approximately 30 millimeters mercury. In addition, blood pressure rises immediately after bleeding.
Induction representative laser doppler flow meter values after bleeding induction show a dramatic decrease in cerebral perfusion reperfusion to an individually different level occurs within five minutes after the insult. In this example, blood distribution along brain supplying arteries and cerebellar fissures are observed. Three hours after the hemorrhage, red lines indicate blood distribution along brain supplying arteries.
The side ipsilateral to the hemorrhage was covered with more blood than the contralateral hemisphere seen. Here is the survival curve following SAH in 49 male, C 57 BL six mice. While attempting this procedure, it's important to monitor the physiologic conditions to secure a reproducible amount of bleeding After its development.
This technique paved the way for researchers in the field of subretinal hemorrhage to explore the path mechanisms of cerebral ischemia early after the insult.
