The overall goal of this procedure is the preparation of a lateral cranial window combined with permanent middle cerebral artery occlusion. To facilitate longitudinal intravital microscopy in mice. This method can help to investigate and answer key questions in the field of cerebellar ischemia research.

The main advantage of this technique is that it offers in vivo imaging of acute and chronic changes after stroke. Begin by administering anesthesia according to an approved animal protocol. Ensure that a surgical plane of anesthesia has been reached by administering a pain stimulus, such as a toe pinch, and noting the absence of a reaction.

Next, use a rodent shaver to remove the fur from the neck to the eyes. Fix the head of the mouse in a Stereotaxic frame. Then cover the eyes with ophthalmic ointment to prevent dehydration.

Clean the shaved area to remove all hairs, and sterilize the scalp with three applications of 74.1%ethanol and 10%tube Propranolol. Using a sterile scalpel, make a midline incision from the neck to the eyes. Then span the skin flap with four tenting sutures.

Next, carefully scrape the periosteum on the left side of the skull. And remove it to the point where the temporal muscle begins. Now use a micro drill to thin the skull at the rim of the bone flap.

To create a four-millimeter diameter frontoparietal craniotomy. Apply saline solution while drilling to avoid heat injury. Once drilled, elevate the bone flap with cannulas and remove it using micro forceps.

Irrigate the area carefully and extensively with saline solution. Mix the dental glue until it has the proper consistency. Then apply it to the bone around the craniotomy.

Place a six-millimeter diameter cover glass on the prepared glue. And fix it with the rest of the dental glue. Irrigation accelerates the curing process and ensures the seal is water-tight.

Probe the dental glue with forceps to ensure that it is dried and hard. After the glue has cured, remove the tenting sutures and close the wound with skin sutures. Administer 0.5 milliliters of sterile saline subcutaneously for fluid balance.

After surgery, monitor the animal in a heated recovery cage until recovered from anesthesia. Ensure that the animal is completely recovered before returning it to a cage with other animals. Always administer analgesia after surgery.

Here, 10 milligrams per milliliter acetaminophen is added to the drinking water. Always provide mashed animal food in a petri dish placed on the floor to make eating simple and to avoid critical weight loss after surgery. Intravital microscopy can be performed on the first day after cranial window preparation.

Apply isoflurane anesthesia according to an approved protocol. Fix the animal in a head-holder and ensure the nose cone is properly fitted. Open the skin suture and clean the window with cotton buds and sterile saline.

After 24 hours, the cranial window should be filled with cerebrospinal fluid, which enables imaging. Perform the middle cerebral artery occlusion procedure five days after cranial window preparation, when the immune response to the procedure has receded. Here, an overview after craniotomy for MCAO induction is shown.

The intraoperative photo shows the vessels after first and second bipolar contact. The distal MCA appears completely occluded. This picture provides a final overview using lower magnification.

After inducing surgical anesthesia with isoflurane as before, shave the fur in the area between the eye and ear, apply ophthalmic ointment to both eyes, and disinfect the skin and surrounding fur with alcohol solution, as before. Place the mouse on a feedback-controlled heating pad to maintain body temperature. Place the animal in the lateral position and fix the nose in the anesthesia mask.

Gently separate the skin of the incision made for the cranial window preparation and identify the temporal muscle underneath. Next, adjust the energy of the high frequency generator and use the bipolar mode. Use electrocoagulation forceps to carefully lift the temporal muscle from the skull, creating a muscle flap without totally removing the muscle.

Then identify the middle cerebral artery below the transparent skull in the rostral part of the temporal area. Dorsal to the retro-orbital sinus. Thin the skull above the MCA branch with a micro drill, while continuously irrigating to avoid heat damage.

Then lift the bone and remove it with micro forceps. Decrease the energy of the high frequency generator to three to five watt. Then, approach the artery from above and gently touch it with the bipolar forceps on both sides without lifting the vessel.

Wait for thirty seconds, and then touch the artery gently with the tip of the bipolar forceps to ensure that the blood flow is permanently interrupted. After MCA occlusion, fix the temporal muscle with one or two stitches at the muscle insertion to cover the bone defect. Then, suture the wound and place the animal into the heated cage until recovered.

This MRI was performed 24 hours after sham surgery, and does not show any lesioned cortical tissue. Whereas this MRI, performed 24 hours after ischemia, clearly depicts the ischemic lesion indicated by an asterisk, located underneath the chronic cranial window. This image shows intravital epifluorescense imaging and two-photon imaging of the cortical vasculature to monitor changes in microcirculation.

Infarct volume assessed via MRI at 24 and 96 hours after ischemia, shows a mean lesion volume of 13.16 millimeters cubed at 24 hours, and 12.2 millimeters cubed at 96 hours. Once mastered, the cranial window preparation can be done in about 30 minutes. And the MCAO in about 15 minutes.

Following this procedure, other methods, like MRI, or dual-photon microscopy can be performed to answer additional questions. These new techniques allow investigators to investigate a neurovascular unit at a molecular and cellular level. And most particularly, in the living brain.